Nucleic acid probes and methods for detecting legionella

ABSTRACT

A method for preparing probes, as well as several probes for use in qualitative or quantitative hybridization assays are disclosed. The method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA selected to be unique to a non-viral organism or group of non-viral organisms sought to-be detected, said region of rRNA being selected by comparing one or more variable region rRNA sequences of said non-viral organism or group of non-viral organisms with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished. Hybridization assay probes for Mycobacterium avium, Mycobacterium intracellulare, the Mycobacterium tuberculosis-complex bacteria, Mycoplasma pneumoniae, Legionella, Salmonella, Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas group I, Neisseria gonorrhoeae, bacteria, and fungi also are disclosed.

The present application is a divisional of Hogan et al., U.S.application Ser. No. 08/200,866, filed Feb. 22, 1994, now U.S. Pat. No.5,541,308, which is file wrapper continuation of Hogan et al., U.S.application Ser. No. 07/806,929, filed Dec. 11, 1991, now abandoned,which is a file wrapper continuation of Hogan et al., U.S. Ser. No.07/295,208, filed Dec. 9, 1988, now abandoned, which was the Nationalfiling of PCT/US87/03009, filed Nov. 24, 1987, which is acontinuation-in-part of Hogan et al., U.S. application Ser. No.07/083,542, filed Aug. 7, 1987, now abandoned, which is acontinuation-in-part of Hogan et al., U.S. Ser. No. 06/934,244, filedNov. 24, 1986, now abandoned, the entirely of each of these priorapplications including drawings are hereby incorporated by referenceherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The inventions described and claimed herein relate to probes and assaysbased on the use of genetic material such as RNA. More particularly, theinventions relate to the design and construction of nucleic acid probesand hybridization of such probes to genetic material of target non-viralorganisms in assays for detection and/or quantitation thereof in testsamples of, e.g., sputum, urine, blood and tissue sections, food, soiland water.

2. Introduction

Two single strands of nucleic acid, comprised of nucleotides, mayassociate ("hybridize") to form a double helical structure in which thetwo polynucleotide chains running in opposite directions are heldtogether by hydrogen bonds (a weak form of chemical bond) between pairsof matched, centrally located compounds known as "bases." Generally, inthe double helical structure of nucleic acids, for example, the baseadenine (A) is hydrogen bonded to the base thymine (T) or uracil (U)while the base guanine (G) is hydrogen bonded to the base cytosine (C).At any point along the chain, therefore, one may find the base pairs ATor AU, TA or UA, GC, or CG. One may also find AG and GU base pairs inaddition to the traditional ("canonical") base pairs. Assuming that afirst single strand of nucleic acid is sufficiently complementary to asecond and that the two are brought together under conditions which willpromote their hybridization, double stranded nucleic acid will result.Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids maybe formed.

Broadly, there are two basic nucleic acid hybridization procedures. Inone, known as "in solution" hybridization, both a "probe" nucleic acidsequence and nucleic acid molecules from a test sample are free insolution. In the other method, the sample nucleic acid is usuallyimmobilized on a solid support and the probe sequence is free insolution.

A probe may be a single strand nucleic acid sequence which iscomplementary in some particular degree to the nucleic acid sequencessought to be detected ("target sequences"). It may also be labelled. Abackground description of the use of nucleic acid hybridization as aprocedure for the detection of particular nucleic acid sequences isdescribed in U.S. application Ser. No. 456,729, entitled "Method forDetection, Identification and Quantitation of Non-Vital Organisms,"filed Jan. 10, 1983 (Kohne I, now issued as U.S. Pat. No. 4,851,330),and U.S. application Ser. No. 655,365, entitled "Method For Detecting,Identifying and Quantitating Organisms and Viruses," filed Sep. 4, 1984(Kohne II, now issued as U.S. Pat. No. 5,288,611), both of which areincorporated by reference, together with all other applications citedherein.

Also described in those applications are methods for determining thepresence of RNA-containing organisms in a sample which might containsuch organisms, comprising the steps of bringing together any nucleicacids from a sample and a probe comprising nucleic acid molecules whichare shorter than the rRNA subunit sequence from which it was derived andwhich are sufficiently complementary to hybridize to the rRNA of one ormore non-viral organisms or groups of non-viral organisms, incubatingthe mixture under specified hybridization conditions, and assaying theresulting mixture for hybridization of the probe and any test samplerRNA. The invention is described to include using a probe which detectsonly rRNA subunit subsequences which are the same or sufficientlysimilar in particular organisms or groups of organisms and is said todetect the presence or absence of any one or more of those particularorganisms in a sample, even in the presence of many non-relatedorganisms.

We have discovered and describe herein a novel method and means fordesigning end constructing DNA probes for use in detecting unique rRNAsequences in an assay for the detection and/or quantitation of any groupof non-viral organisms. Some of the inventive probes herein may be usedto detect and/or quantify a single species or strain of non-viralorganism and others may be used to detect and/or quantify members of anentire genus or desired phylogenetic grouping.

SUMMARY OF THE INVENTION

In a method of probe preparation and use, a single stranddeoxyoligonucleotide of particular seguence and defined length is usedin a hybridization assay to determine the presence or amount of rRNAfrom particular target non-viral organisms to distinguish them fromtheir known closest phylogenetic neighbors. Probe sequences which arespecific, respectively, for 16S rRNA variable subsequences ofMycobacterium avium, Mycobacterium intracellulare and the Mycobacteriumtuberculosis-complex bacteria, and which do not cross react with nucleicacids from each other, or any other bacterial species or respiratoryinfectious agent, under proper stringency, are described and claimed. Aprobe specific to three 23S rRNA variable region subsequences from theMycobacterium tuberculosis-complex bacteria is also described andclaimed, as are rRNA variable region probes useful in hybridizationassays for the genus Mycobacterium (16S 23S rRNA specific), Mycoplasmapneumoniae (5S and 16S rRNA-specific), Chlamydia trachomatis (16S and23S rRNA specific), Enterobacter cloacae (23S rRNA specific),Escherichia coli (16S rRNA specific), Legionella (16S and 23S rRNAspecific), Salmonella (16S and 23S rRNA specific), Enterococci (16S rRNAspecific), Neisseria gonorrhoeae (16S rRNA specific), Campylobacter (16SrRNA specific), Proteus mirabilis (23S rRNA specific), Pseudomonas (23SrRNA specific), fungi (18S and 28S rRNA specific), and bacteria (16S and23S rRNA specific).

In one embodiment of the assay method, a test sample is first subjectedto conditions which release rRNA from any non-viral organisms present inthat sample. rRNA is single stranded and therefore available forhybridization with sufficiently complementary genetic material once soreleased. Contact between a probe, which can be labelled, and the rRNAtarget may be carried out in solution under conditions which promotehybridization between the two strands. The reaction mixture is thenassayed for the presence of hybridized probe. Numerous advantages of thepresent method for the detection of non-viral organisms over prior arttechniques, including accuracy, simplicity, economy and speed willappear more fully from the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a chart of the primary structure of bacterial 16S rRNA forEscherichia coli, depicting standard reference numbers for bases.

FIG. 2 is a chart of the primary structure of bacterial 23S rRNA forEscherichia coli, depicting standard reference numbers for bases.

FIG. 3 is a chart of the primary structure of bacterial 5S rRNA forEscherichia coli, depicting standard reference numbers for bases.

FIG. 4 is a chart of the primary structure for the 18S rRNA forSaccharomyces cerevisiae, depicting standard reference numbers forbases.

FIG. 5 is a chart of the primary structure for the 28S rRNA forSaccharomyces cerevisiae, depicting standard reference numbers forbases.

FIG. 6 is a diagram showing the locations in the 16S rRNA (using E. colireference numbers) which differ between 12 different sets of relatedorganisms. In Example 1, for example, 99.7% refers to the difference in16S rRNA between Clostridium botuliniumg and Clostridium subterminale.

FIG. 7 is a diagram showing the locations in the first 1500 bases of 23SrRNA (using E. coli reference numbers) which differ between 12 differentsets of related organisms.

FIG. 8 is a diagram showing the locations in the terminal bases of 23SrRNA (using E. coli reference numbers) which differ between 12 differentsets of related organisms.

FIG. 9 is a schematic representation of the location of probes capableof hybridizing to the 16S rRNA.

FIG. 10 is a schematic representation of the location of probes capableof hybridizing to the first 1500 bases of the 23S rRNA.

FIG. 11 is a schematic representation of the location of probes capableof hybridizing to the terminal bases of 23S rRNA.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following terms, as used in this disclosure and claims, are definedas:

nucleotide: a subunit of a nucleic acid consisting of a phosphate group,a 5' carbon sugar and a nitrogen containing base. In RNA the 5' carbonsugar is ribose. In DNA, it is a 2-deoxyribose. The term also includesanalogs of such subunits.

nucleotide polymer: at least two nucleotides linked by phosphodiesterbonds.

oligonucleotide: a nucleotide polymer generally about 10 to about 100nucleotides in length, but which may be greater than 100 nucleotide inlength.

nucleic acid probe: a single stranded nucleic acid sequence that willcombine with a complementary single stranded target nucleic acidsequence to form a double-stranded molecule (hybrid). A nucleic acidprobe may be an oligonucleotide or a nucleotide polymer.

hybrid: the complex formed between two single stranded nucleic acidsequences by Watson-Crick base pairings or non-canonical base pairingsbetween the complementary bases.

hybridization: the process by which two complementary strands of nucleicacids combine to form double stranded molecules (hybrids).

complementarity: a property conferred by the base sequence of a singlestrand of DNA or RNA which may form a hybrid or double stranded DNA:DNA,RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick basepairs on the respective strands. Adenine (A) usually complements thymine(T) or Uracil (U), while guanine (G) usually complements cytosine (C).

stringency: term used to describe the temperature and solventcomposition existing during hybridization and the subsequent processingsteps. Under high stringency conditions only highly homologous nucleicacid hybrids will form; hybrids without a sufficient degree ofcomplementarity will not form. Accordingly, the stringency of the assayconditions determine the amount of complementarity needed. between twonucleic acid strands forming a hybrid. Stringency is chosen to maximizethe difference in stability between the hybrid formed with the targetand the nontarget nucleic acid.

probe specificity: characteristic of a probe which describes its abilityto distinguish between target and non-target sequences. Dependent onsequence and assay conditions. Probe specificity may be absolute (i.e.,probe able to distinguish between target organisms and any nontargetorganisms), or it may be functional (i.e., probe able to distinguishbetween the target organism and any other organism normally present in aparticular sample). Many probe sequences can be used for either broad ornarrow specificity depending on the conditions of use.

variable region: nucleotide polymer which differs by at least one basebetween the target organism and nontarget organisms contained in asample.

conserved region: a region which is not variable.

sequence divergence: process by which nucleotide polymers become lesssimilar during evolution.

sequence convergence: process by which nucleotide polymers become moresimilar during evolution.

bacteria: members of the phylogenetic group eubacteria, which isconsidered one of the three primary kingdoms.

Tm: temperature at which 50% of the probe is converted from thehybridized to the unhybridized form.

thermal stability: Temperature at which 50% of the probe:target hybridsare converted to the single stranded form. Factors which affect thethermal stability can affect probe specificity and therefore, must becontrolled. Whether a probe sequence is useful to detect only a specifictype of organism depends largely on the thermal stability differencebetween probe:target hybrids ("P:T") and probe:nontarget hybrids("P:NT"). In designing probes the Tm P:T minus the Tm P:NT should be aslarge as possible.

In addition to a novel method for selecting probe sequences, we havediscovered that it is possible to create a DNA probe complementary to aparticular rRNA sequence obtained from a single type of targetmicroorganism and to successfully use that probe in a non-cross reactingassay for the detection of that single microorganism, even in thepresence of its known, most closely related taxonomic or phylogeneticneighbors. With the exception of viruses, all prokaryctic organismscontain rRNA molecules including 5S rRNA, 16S rRNA, and a larger rRNAmolecule known as 23S rRNA. Eukaryotes are known to have 5.0S, 5.8S, 18Sand 28S rRNA molecules or analogous structures. (The term "16S like"sometimes is used to refer to the rRNA found in the small ribosomalsubunit, including 18S and 17S rRNA. Likewise the term "23S like" rRNAsometimes is used to refer to the rRNA found in the large ribosomalsubunit. 5.8S rRNA is equivalent to the 5' end of the 23S like rRNA.)These rRNA molecules contain nucleotide sequences which are highlyconserved among all organisms thus far examined. There are known methodswhich allow a significant portion of these rRNA sequences to bedetermined. For example, complementary oligonucleotide primers of about20-30 bases in length can be hybridized to universally conserved regionsin purified rRNA that are specific to the 5S, 16S, or 23S subunits andextended with the enzyme reverse transcriptase. Chemical degradation ordideoxynucleotide-terminated sequencing reactions can be used todetermine the nucleotide sequence of the extended product. Lane, D. J.et al., Proc. Nat'l Acad. Sci. USA 82, 6955-6959 (1985).

In our invention, comparison of one or more sequenced rRNA variableregions from a target organism to one or more rRNA variable regionsequences from a closely related bacterial species is utilized to selecta sequence unique to the rRNA of the target organism. rRNA is preferableto DNA as a probe target because of its relative abundance and stabilityin the cell and because of its patterns of phylogenetic conservation.

Notwithstanding the highly conserved nature of rRNA, we have discoveredthat a number of regions of the rRNA molecule which can vary insequence, can vary even between closely related species and can,therefore, be utilized to distinguish between such organisms.Differences in the rRNA molecule are not distributed randomly across theentire molecule, but rather are clustered into specific regions. Thedegree of conservation also varies, creating a unique pattern ofconservation across the ribosomal RNA subunits. The degree of variationand the distribution thereof, can be analyzed to locate target sites fordiagnostic probes. This method of probe selection may be used to selectmore than one sequence which is unique to the rRNA of a target organism.

We have identified variable regions by comparative analysis of rRNAsequences both published in the literature and sequences which we havedetermined ourselves using procedures known in the art. We use a SunMicrosystems (TM) computer for comparative analysis. The compiler iscapable of manipulating many sequences of data at the same time.Computers of this type and computer programs which may be used oradapted for the purposes herein disclosed are commercially available.

Generally, only a few regions are useful for distinguishing betweenclosely related species phylogenetically conserved genus, for example,the region 400-500 bases from the 5' end of the 16S rRNA molecule. Ananalysis of closely related organisms (FIGS. 6, 7 and 8) reveals thespecific positions (variable regions) which vary between closely relatedorganisms. These variable regions of rRNA molecules are the likelycandidates for probe design.

FIGS. 6, 7 and 8 display the variations in 16S and 23S rRNA's betweentwo different bacteria with decreasing amounts of similarity betweenthem. Closer analysis of these figures reveals some subtle patternsbetween these closely related organisms. In all cases studied, we haveseen sufficient variation between the target organism and the closestphylogenetic relative found in the same sample to design the probe ofinterest. Moreover, in all cases studied to date, the per centsimilarity between the target organism (or organisms) and the closestphylogenetically related organisms found in the same sample has beenbetween 90% and 99%. Interestingly, there was enough variation evenbetween the rRNA's of Neisseria's gonorrhoeae and meningitidis (SeeExample 21) to design probes--despite the fact that DNA:DNA homologystudies suggested these two species might actually be one and the same.

These figures also show that the differences are distributed across theentire 16S and 23S rRNA's. Many of the differences, nonetheless, clusterinto a few regions. These locations in the rRNA are good candidates forprobe design, with our current assay conditions. We also note that thelocations of these increased variation densities usually are situated inthe same regions of the 16S and 23S rRNA for comparable per centsimilarity values. In this manner, we have observed that certain regionsof the 16S and 23S rRNA are the most likely sites in which significantvariation exists between the target organism and the closestphylogenetic relatives found in a sample. We have disclosed and claimedspecies specific probes which hybridize in these regions of significantvariation between the target organism and the closest phylogeneticrelative found in a sample.

FIGS. 9, 10 and 11 are a schematic representation of the location ofprobes disclosed and claimed herein. Because 16S and 23S RNAs do not, asa rule, contain sequences of duplication longer than about sixnucleotides in length, probes designed by these methods are specific toone or a few positions on the target nucleic acid.

The sequence evolution at each of the variable regions (for example,spanning a minimum of 10 nucleotides) is, for the most part divergent,not convergent. Thus, we can confidently design probes based on a fewrRNA sequences which differ between the target organism and itsphylogenetically closest relatives. Biological and structuralconstraints on the rRNA molecule which maintain homologous primary,secondary and tertiary structure throughout evolution, and theapplication of such constraints to probe diagnostics is the subject ofongoing study. The greater the evolutionary distance between organisms,the greater the number of variable regions which may be used todistinguish the organisms.

Once the variable regions are identified, the sequences are aligned toreveal areas of maximum homology or "match". At this point, thesequences are examined to identify potential probe regions. Twoimportant objectives in designing a probe are to maximize homology tothe target sequence(s) (greater than 90% homology is recommended) and tominimize homology to non-target sequence(s) (less than 90% homology tonontargets is recommended). We have identified the following usefulguidelines for designing probes with desired characteristics.

First, probes should be positioned so as to minimize the stability ofthe probe:nontarget nucleic acid hybrid. This may be accomplished byminimizing the length of perfect complementarity to non-targetorganisms, avoiding G and C rich regions of homology to non-targetsequences, and by positioning the probe to span as many destabalizingmismatches as possible (for example, dG:rU base pairs are lessdestabalizing than some others).

Second, the stability of the probe: target nucleic acid hybrid should bemaximized. This may be accomplished by avoiding long A and T richsequences, by terminating the hybrids with G:C base pairs and bydesigning the probe with an appropriate Tm. The beginning and end pointsof the probe should be chosen so that the length and % G and % C resultin a Tm about 2°-10° C. higher than the temperature at which the finalassay will be performed. The importance and effect of various assayconditions will be explained further herein. Third, regions of the rRNAwhich are known to form strong structures inhibitory to hybridizationare less preferred. Finally, probes with extensive self-complementarityshould be avoided.

In some cases, there may be several sequences from a particular regionwhich will yield probes with the desired hybridization characteristics.In other cases, one sequence may be significantly better than anotherwhich differs merely by a single base.

The following chart indicates how, for one embodiment of the inventionuseful in the detection of a nucleic acid in the presence of closelyrelated nucleic acid sequences, unique sequences can be selected. Inthis example, rRNA sequences have been determined for organisms A-E andtheir sequences, represented numerically, are aligned as shown. It isseen that sequence 1 is common to all organisms A-E. Sequences 2-6 arefound only in organisms A, B and C, while sequences 8, 9 and 10 areunique to organism A. Therefore, a probe complementary to sequences 8, 9or 10 would specifically hybridize to organism A.

    ______________________________________                                        Illustrative Pattern of Sequence                                              Relationships Among Related Bacteria                                          Organism                                                                             rRNA Sequence                                                          ______________________________________                                        A      1      2     3    4    5   6    7   8    9   10                        B      1      2     3    4    5   6    7   11   12  13                        C      1      2     3    4    5   6    14  15   16  17                        D      1      18    19   20   21  22   23  24   25  26                        E      1      18    19   20   21  27   28  29   30  31                        ______________________________________                                    

In cases where the patterns of variation of a macromolecule are known,for example, rRNA, one can focus on specific regions as likelycandidates for probe design. However, it is not always necessary todetermine the entire nucleic acid sequence in order to obtain a probesequence. Extension from any single oligonucleotide primer can yield upto 300-400 bases of sequence. When a single primer is used to partiallysequence the rRNA of the target organism and organisms closely relatedto the target, an alignment can be made as outlined above. Plainly, if auseful probe sequence is found, it is not necessary to continue rRNAsequencing using other primers. If, on the other hand, no useful probesequence is obtained from sequencing with a first primer, or if highersensitivity is desired, other primers can be used to obtain moresequences. In those cases where patterns of variation for a molecule arenot well understood, more sequence data may be required prior to probedesign.

Thus, in Examples 1-3 below, two 16S-derived primers were used. Thefirst primer did not yield probe sequences which met the criteria listedherein. The second primer yielded probe sequences which were determinedto be useful following characterization and testing for specificity asdescribed. In Example 4, six 23S primers were used prior to locating theprobe sequence set forth.

Once a presumptive unique sequence has been identified, a complementaryDNA oligonucleotide is synthesized. This single stranded oligonucleotidewill serve as the probe in the DNA/rRNA assay hybridization reaction.Defined oligonucleotides may be synthesized by any of several well knownmethods, including automated solid-phase chemical synthesis usingcyano-ethylphosphoramidite precursors. Barons, A. D. et al., NucleicAcids Research, 12, 4051-4060 (1984). In this method,deoxyoligonucleotides are synthesized on solid polymer supports. Releaseof the oligonucleotide from the support is accomplished by treatmentwith ammonium hydroxide at 60° C. for 16 hours. The solution is driedand the crude product is dissolved in water and separated onpolyacrylamide gels which generally may vary from 10-20% depending uponthe length of the fragment. The major band, which is visualized byultraviolet back lighting, is cut from the gel with a razor blade andextracted with 0.1M ammonium acetate, pH 7.0, at room temperature for8-12 hours. Following centrifugation, the supernatant is filteredthrough a 0.4 micron filter and desalted on a P-10 column (Pharmacia).Other well known methods for construction of synthetic oligonucelotidesmay, of course, be employed.

Current DNA synthesizers can produce large amounts of synthetic DNA.After synthesis, the size of the newly made DNA is examined by gelfiltration and molecules of varying size are generally detected. Some ofthese molecules represent abortive synthesis events which occur duringthe synthesis process. As part of post-synthesis purification, thesynthetic DNA is usually size fractionated and only those moleculeswhich are the proper length are kept. Thus, it is possible to obtain apopulation of synthetic DNA molecules of uniform size.

It has been generally assumed, however, that synthetic DNA is inherentlycomposed of a uniform population of molecules all of the same size andbase sequence, and that the hybridization characteristics of everymolecule in the preparation should be the same. In reality, preparationsof synthetic DNA molecules are heterogeneous and are composed ofsignificant numbers of molecules which, although the same size, are insome way different from each other and have different hybridizationcharacteristics. Even different preparations of the same sequence cansometimes have different hybridization characteristics.

Accordingly, preparations of the same synthetic probe sequence can havedifferent hybridization chacteristics. Because of this the specificityof probe molecules from different preparations can be different. Thehybridization characteristics of each preparation should be examined inorder to determine the hybridization conditions which must be used inorder to obtain the desired probe specificity. For example, thesynthetic probe described in Example 4 below has the specificity profiledescribed in Table 14. This data was obtained by using the hybridizationand assay conditions described. A separate preparation of this probewhich has different hybridization characteristics may not have preciselythe same specificity profile when assayed under the conditions presentedin Example 4. Such probe preparations have been made. To obtain thedesired specificity, these probes can be hybridized and assayed underdifferent conditions, including salt concentration and/or temperature.The actual conditions under which the probe is to be used must bedetermined, or matched to extant requirements, for each batch of probesince the art of DNA synthesis is somewhat imperfect.

Following synthesis and purification of a particular oligonucleotidesequence, several procedures may be utilized to determine theacceptability of the final product. The first is polyacrylamide gelelectrophoresis, which is used to determine size. The oligonucleotide islabelled using, for example, ³² P-ATP and T₄ polynucleotide kinase. Thelabelled probe is precipitated in ethanol, centrifuged and the driedpellet resuspended in loading buffer (80% formamide, 20 mM NaOH, 1 mMEDTA, 0.1% bromophenol blue and 0.1% xylene cyanol). The samples areheated for five minutes at 90° C. and loaded onto a denaturingpolyacrylamide gel. Electrophoresis is carried out in TBE buffer (0.1MTris HCl pH 8.3, 0.08M boric acid, 0.002M EDTA) for 1-2 hours at 1,000volts. Following electrophoresis of the oligonucleotide the gel isexposed to X-ray film. The size of the oligonucleotide is then computedfrom the migration of oligonucleotide standards run concurrently.

The sequence of the synthetic oligonucleotide may also be checked bylabelling it at the 5' end with ³² P-ATP and T₄ polynucleotide kinase,subjecting it to standard chemical degradation techniques, Maxam, A. M.and Gilbert, W., Proc. Nat'l. Acad. Sci. USA 74, 560-564 (1980), andanalyzing the products on polyacrylamide gels. Preferably, thenucleotide sequence of the probe is perfectly complementary to thepreviously identified unique rRNA sequence, although it need not be.

The melting profile, including the melting temperature (Tm) of theoligonucleotide/rRNA hybrids should also be determined. One way todetermine Tm is to hybridize a ³² P-labelled oligonucleotide to itscomplementary target nucleic acid at 50° C. in 0.1M phosphate buffer, pH6.8. The hybridization mixture is diluted and passed over a 2 cmhydroxyapatite column at 50° C. The column is washed with 0.1M phosphatebuffer, 0.02% SDS to elute all unhybridized, single-stranded probes. Thecolumn temperature is then dropped 15° C. and increased in 5° C.increments until all of the probe is single-stranded. At eachtemperature, unhybridized probe is eluted and the counts per minute(cpm) in each fraction determined. The number of cpm shown to be boundto the hydroxyapatite divided by the total cpm added to the columnequals the percent hybridization of the probe to the target nucleicacid.

An alternate method for determining thermal stability of a hybrid isoutlined below. An aliquot of hybrid nucleic acid is diluted into 1 mlof either 0.12M phosphate buffer, 0.2% SDS, 1 mM EDTA, 1 mM EGTA or anappropriate hybridization buffer. Heat this 1 ml of solution to 45° C.for 5 minutes and place it into a room temperature water bath to coolfor 5 minutes. Assay this 1 ml of hybrid containing solution over ahydroxyapatite column, capturing the hybrid and washing away unboundprobe. If a hybridization solution other than the 0.12M phosphate bufferis used, then a dilution of the hybridization solution into the 0.12Mphosphate buffer will be necessary for binding. Keep taking aliquots ofhybrid and diluting into 1 ml of hybridization solution or into thestandard 0.12M phosphate buffer solution described above while raisingthe heating temperature 5° C. at a time. Continue this until all of thehybrid is dissociated. The point where one half of the hybrid isconverted to the dissociated form is considered the Tm. The Tm for agiven hybrid will vary depending on the hybridization solution beingused because the thermal stability depends upon the concentration ofdifferent salts, detergents, and other solutes which effect relativehybrid stability during thermal denaturation.

Because the extent and specificity of hybridization reactions such asthose described herein are affected by a number of factors, manipulationof one or more of those factors will determine the exact sensitivity andspecificity of a particular probe, whether perfectly complementary toits target or not. For example, the base composition of the probe may besignificant because G-C base pairs exhibit greater thermal stability ascompared to A-T base pairs due to additional hydrogen bonding. Thus,hybridization involving complementary nucleic acids of higher G-Ccontent will be stable at higher temperatures.

We have discovered that the length of the target nucleic acid sequenceand, accordingly, the length of the probe sequence can also beimportant. While it is possible for nucleic acids that are not perfectlycomplementary to hybridize, the longest stretch of perfectly homologousbase sequence will normally primarily determine hybrid stability. Whileoligonucleotide probes of different lengths and base composition may beused, oligonucleotide probes preferred in this invention are betweenabout 15 and about 50 bases in length and are at least about 75-100%homologous to the target nucleic acid. For most applications 95-100%homology to the target nucleic acid is preferred.

Ionic strength and incubation temperature should also be taken intoaccount in constructing a probe. It is known that the rate ofhybridization will increase as ionic strength of the reaction mixtureincreases and that the thermal stability of hybrids will increase withincreasing ionic strength. In general, optimal hybridization forsynthetic oligonucleotide probes of about 15-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

As to nucleic acid concentration, it is known that the rate ofhybridization is proportional to the concentration of the twointeracting nucleic acid species. Thus, the presence of compounds suchas dextran and dextran sulphate are thought to increase the localconcentration of nucleic acid species and thereby result in an increasedrate of hybridization. Other agents which will result in increased ratesof hybridization are specified in U.S. application Ser. No. 627,795,entitled "Accelerated Nucleic Acid Reassociation Method", filed Jul. 5,1984, Continuation-in-Part thereof, Serial No. (net yet assigned), filedJun. 4, 1987, and U.S. application Ser. No. 816,711, entitled"Accelerated Nucleic Acid Reassociation Method", filed Jan. 7, 1986,both of which are incorporated by reference. (U.S. application Ser. No.07/644,879, which is a continuation of U.S. application Ser. No.816,711, issued as U.S. Pat. No. 5,132,207on Jul. 21, 1992.) On theother hand, chemical reagents which disrupt hydrogen bonds such asformamide, urea, DMSO, and alcohols will increase the stringency ofhybridization.

Selected oligonucleotide probes may be labelled by any of several wellknown methods. Useful labels include radioisotopes as well asnon-radioactive reporting groups. Isotopic labels include ³ H, ³⁵ S, ³²P, ¹²⁵ I, Cobalt and ¹⁴ C. Most methods of isotopic labelling involvethe use of enzymes and include the known methods of nick translation,end labelling, second strand synthesis, and reverse transcription. Whenusing radio-labelled probes, hybridization can be detected byautoradiography, scintillation counting, or gamma counting. Thedetection method selected will depend upon the hybridization conditionsand the particular radioisotope used for labelling.

Non-isotopic materials can also be used for labelling, and may beintroduced by the incorporation of modified nucleotides through the useof enzymes or by chemical modification of the probe, for example, by theuse of non-nucleotide linker groups. Non-isotopic labels includefluorescent molecules, chemiluminescent molecules, enzymes, cofactors,enzyme substrates, haptens or other ligands. We currently prefer to useacridinium esters.

In one embodiment of the DNA/rRNA hybridization assay invention, alabelled probe and bacterial target nucleic acids are reacted insolution. rRNA may be released from bacterial cells by the sonicdisruption method described in Murphy, K. A. et al., U.S. applicationSer. No. 841,860, entitled "Method for Releasing RNA and DNA FromCells", filed Mar. 20, 1986, which is incorporated herein by reference.(U.S. application Ser. No. 07/711,114, which is a continuation of U.S.application Ser. No. 07/298,765, which is a continuation of U.S.application Ser. No. 06/641,860, issued as U.S. Pat. No. 5,374,522, onJan. 20, 1994.) Other known methods for disrupting cells include the useof enzymes, osmotic shock, chemical treatment, and vortexing with glassbeads. Following or concurrent with the release of rRNA, labelled probemay be added in the presence of accelerating agents and incubated at theoptimal hybridization temperature for a period of time necessary toachieve significant reaction. Following this incubation period,hydroxyapatite may be added to the reaction mixture to separate theprobe/rRNA hybrids from the non-hybridized probe molecules. Thehydroxyapatite pellet is washed, recentrifuged and hybrids detected bymeans according to the label used.

Twenty-one embodiments illustrative of the claimed inventions are setforth below, in which a synthetic probe or probes complementary to aunique rRNA sequence from a target organism, or group of organisms isdetermined, constructed and used in a hybridization assay.

DESCRIPTION OF PARTICULAR EMBODIMENTS

Mycobacterium are acid-fast, alcohol fast, aerobic, non-mobile bacilli.Their lipid content is high and their growth slow. Mycobacterium aviumand Mycobacterium intracellulare are together referred to as M.avium-intracellulare because they are so difficult to differentiate.Recently, the M. avium complex, which includes M. intracellulare, wasshown to be the second most commonly isolated, clinically significantMycobacterium. Good, R. C. et al., J. Infect. Dis, 146, 829-833 (1982).More recent evidence indicates that these organisms are a common causeof opportunistic infection in patients with AIDS (acquired immunedeficiency syndrome). Gill, V. J. et al., J. Clin. Microbio. 22, 543-546(1985). Treatment of such infections in AIDS patients is difficultbecause these organisms are resistant to most antituberculosis drugs.Often a combination of five drugs are used in therapy. The severity ofthese infections also requires rapid diagnosis which, prior to theinvention herein, was not available.

Members of the Mycobacterium tuberculosis complex (Mtb) includeMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanumand Mycobacterium microti. The first three are pathogenic for humanswhile the last is an animal pathogen. These organisms produce slowlydeveloping granulomas on the skin or they may invade internal organs.Tuberculosis of the lungs can be disseminated to other parts of the bodyby the circulatory system, the lymph system, or the intestinal tract.Despite advances in public health and the advent of effectivechemotherapy, Mycobacterial disease, tuberculosis in particular,continues to represent a major world-wide health problem.

The classical method for detecting bacteria in a test sample involvesculturing of the sample in order to expand the number of bacterial cellspresent into observable colony growths which can be identified andenumerated. If desired, the cultures can also be subjected to additionaltesting in order to determine antimicrobial susceptibility. Currently,the most widely used procedures for the detection, isolation andidentification of Mycobacterium species are the acid-fast bacilli (AFB)smear (using either the Ziehl-Neelsen or fluorochrome techniques),culture methods using Lowenstein-Jensen media and Middlebrook media, andbiochemical tests. The AFB relies on the high lipid content ofMycobacterium to retain dye after exposure to acid-alcohol. While theAFB smear test is relatively rapid and simple to perform it does notalways detect Mycobacteria and will not differentiate betweenMycobacterium avium and non-tuberculosis species, between Mycobacteriumintracellulare and non-tuberculosis species, or between Mycobacteriumtuberculosis-complex bacilli and non-tuberculosis species. For accurateidentification of the infecting Mycobacterial species the clinician mustrely on culture results which can require anywhere from 3 to 8 weeks ofgrowth followed by extensive biochemical testing. Other tests have beendeveloped based on the detection of metabolic products fromMycobacterium using carbon-14 labelled substrates. In particular, theBactec (TM) instrument can detect the presence of Mycobacterium within 6to 10 days of the time of innoculation. Gill, V. J., supra. However, thetest does not distinguish Mycobacterium species. It is often importantto make this determination so that particular drugs to which theorganism is susceptible may be prescribed. For traditional culturemethods, this requires an additional 2 to 3 weeks and for the Bactecmethod, an additional 6 to 10 days.

In addition, specific embodiments for Mycoplasma pneumoniae, theMycobacterium, Legionella, Salmonella, Chlamydia trachomatis,Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E.coli, Pseudomonas Group I, bacteria, fungi and Neisseria gonorrhoeae areset forth in the following examples.

As indicated by the below examples, the present invention hassignificant advantages over each of these prior art methods not only inthe enhanced accuracy, specificity and simplicity of the test, but alsoin greatly reducing the time to achieve a diagnosis. The invention makespossible a definitive diagnosis and initiation of effective treatment onthe same day as testing.

EXAMPLE 1

Described below is the preparation of a single stranddeoxyoligonuoleotide of unique sequence and defined length which islabelled and used as a probe in a solution hybridization assay to detectthe presence of rRNA from Mycobacterium avium. This unique sequence isspecific for the rRNA of Mycobacterium avium and does not significantlycross-react under the hybridization conditions of this Example, withnucleic acids from any other bacterial species or respiratory infectiousagent, including the closely-related Mycobacterium intracellulare. Thisprobe is able to distinguish the two species, notwithstanding anapproximate 98% rRNA homology between the two species. In this Example,as well as in Examples 2 and 3, sequences for M. avium, M. tuberculosiscomplex, M. intracellulare and related organisms were obtained by usinga specific primer to a highly conserved region in the 16S rRNA. Thesequence of this primer, derived from E. coli rRNA, was 5'-GGC CGT TACCCC ACC TAC TAG CTA AT-3'. 5 nanograms of primer was mixed with 1microgram of each rRNA to be sequenced in the presence of 0.1M KCl and20 mM Tris-HCl pH 8.3 in a final volume of 10 microliters. The reactionswere heated 10 min. at 45° C. and then placed on ice. 2.5 microliters of³⁵ S dATP and 0.5 microliters of reverse transcriptase were added. Thesample was allquoted into 4 tubes, each tube containing either dideoxyA, G, T, or C. The concentrations of these nucleotides are set forth inLane et al., supra. The samples were incubated at 40° C. for 30 minutes,and were then precipitated in ethanol, centrifuged and the pelletslyophilized dry. Pellets were resuspended in 10 microliters formamidedyes (100% formamide, 0.1% bromphenol blue and 0.1% xylene cyanol), andloaded onto 80 cm 8% polyacrylamide gels. The gels were run at 2000volts for 2-4 hours.

Thus, nucleotide sequences for the 16S rRNA of Mycobacterium avium andwhat were considered to be its closest phylogenetic neighbors,Mycobacterium intracellulare and Mycobacterium tuberculosis, weredetermined by the method of Lane, D. J. et al., Proc. Nat. Acad. Sci.USA 82:6955 (1985). In addition to determining the rRNA sequences forthe organisms noted above, a spectrum of clinically significantMycobacterium were also sequenced. These included M. fortuitum, M.scrofulaceum and M. chelonae. Selected members of several genera closelyrelated to Mycobacterium were also sequenced, including Rhodococousbronchialis, Corynebacterium xerosis and Norcardia asteroides.

Partial rRNA sequences from the above organisms were aligned for maximumnucleotide homology, using commercially available software fromIntelligenetics, Inc., 1975 El Camino Real West, Mountain View, Calif.94040-2216 (IFIND Program). From this alignment, regions of sequenceunique to Mycobacterium avium were determined. The probe was selected sothat it was perfectly complementary to a target nucleic acid sequenceand so that it had a 10% or greater mismatch with the aligned rRNA fromits known closest phylogenetic neighbor. A sequence 38 bases in lengthwas chosen. The number of mismatched bases relative to the Mycobacteriumavium sequence were as follows: Mycobacterium tuberculosis (8);Mycobacterium intracellulare (5); Mycobacterium scrofulaceum (6);Mycobacterium chelonae (12); and Mycobacterium fortuitum (10).

The following cDNA sequence was characterized by the criteria of length,Tm, and sequence analysis as described at pages 7-8 above and wasdetermined to be specific for the rRNA Mycobacterium avium:

    ACCGCAAAAGCTTTCCACCAGAAGACATGCGTCTTGAG.

This sequence is complementary to a unique segment found in the 16S rRNAof Mycobacterium avium. The size of the probe is 38 bases. The probe hasa Tm of 74° C. and sequence analysis by the method of Maxam & Gilbert(1980), supra, confirmed that the probe was correctly synthesized. Theprobe is capable of hybridizing to rRNA of M. avium in the regioncorresponding to bases 185-225 of E. coli 16rRNA.

To demonstrate the reactivity of this sequence for Mycobacterium avium,it was tested as a probe in hybridization reactions under the followingconditions. ³² P-end-labeled oligonucleotide probes were mixed with 1microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacterium avium andreacted in 0.12M PB hybridization buffer (equimolar amounts of Na₂ HPO₄and NaH₂ PO₄), 1 mM EDTA and 0.02% SDS (sodium dodecyl sulfate) at 65°C. for 60 minutes in a final volume of 50 microliters. In separate tubesthe probe was mixed with the hybridization buffer both with and withouttarget present. Following separation on hydroxyapatite as outlined inthe patent applications identified at page 2, supra, the hybrids werequantitated by scintillation counting. These results are presented inTable 1, showing that the probe has a high extent of reaction tohomologous target and very little non-specific binding to thehydroxyapatite.

                  TABLE 1                                                         ______________________________________                                        HYBRIDIZATION OF THE M. AVIUM PROBE                                           TO HOMOLOGOUS TARGET rRNA*                                                                    plus rRNA                                                                              minus rRNA                                           ______________________________________                                        M. avium probe  85-95%   0.5%                                                 ______________________________________                                         ##STR1##                                                                 

Specificity of the probe for M. avium was tested by mixing the ³² Plabeled probe with rRNA released from cells of 29 other species ofmycobacteria by the sonic disruption techniques described in Murphy etal., U.S. Pat. No. 5,374,522. 1×10⁸ cells were suspended in 0.1 ml 5%SDS and sonicated for 10 minutes at 50°-60° C. 1.0 ml of hybridizationbuffer (45% sodium diisobutyl sulfosuccinate, 40 mM phosphate buffer pH6.8 and 1 mM EDTA) was added and the mixture incubated for 60 minutes at72° C. Following incubation, 4.0 ml of hydroxyapatite solution (0.14Msodium phosphate buffer, pH 6.8, 0.02% SDS and 1.0 gram hydroxyapatiteper 50 mls solution) was added and incubated for 5 minutes at 72° C. Thesample was centrifuged and the supernatant removed. 4.0 ml wash solution(0.14M sodium phosphate pH 6.8) was added and sample was vortexed,centrifuged and the supernatant removed. The radioactivity bound to thehydroxyapatite was determined by scintillation counting. The results areshown in Table 2 and indicate that the probe is specific forMycobacterium avium and does not react with any other mycobacterialspecies, including Mycobacterium intracellulare.

                  TABLE 2                                                         ______________________________________                                        HYBRIDIZATION OF THE M. AVIUM PROBE                                           TO MYCOBACTERIAL SPECIES                                                      Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Mycobacterium africanum                                                                         25420   1.0                                                 M. asiaticum      25276   1.2                                                 M. avium          25291   87.6                                                M. bovis          19210   1.2                                                 M. bovis (BCG)    19015   1.0                                                 M. chelonae       14472   0.9                                                 M. flavescens     14474   0.9                                                 M. fortuitum      6841    1.0                                                 M. gastri         15754   1.2                                                 M. gordonae       14470   1.2                                                 M. haemophilum    29548   1.3                                                 M. intracallulare 13950   1.5                                                 M. kansasii       12478   1.2                                                 M. malmoense      29571   1.2                                                 M. marinum        827     1.2                                                 M. nonchromogenicum                                                                             1930    1.1                                                 M. phlei          11758   1.3                                                 M. scrofulaceum   19981   1.2                                                 M. shimoidei      27962   2.3                                                 M. simiae         25275   1.2                                                 M. smegmatis      e14468  1.0                                                 M. szulgai        23069   1.0                                                 M. terrae         15755   1.2                                                 M. thermoresistibile                                                                            19527   1.3                                                 M. triviale       23292   1.2                                                 M. tuberculosis (avirulent)                                                                     25177   1.4                                                 M. tuberculosis (virulent)                                                                      27294   1.1                                                 M. ulcerans       19423   1.4                                                 M. vaccae         15483   1.2                                                 M. xenopi         19971   1.5                                                 ______________________________________                                    

As shown in Table 3 the probe also did not react with the rRNA from anyof the respiratory pathogens which were also tested by the method justdescribed. Nor did the probe react with any other closely related orphylogenetically more diverse species of bacteria also tested by thatmethod (Table 4).

                  TABLE 3                                                         ______________________________________                                        HYBRIDIZATION OF M. AVIUM PROBE TO                                            RESPIRATORY PATHOGENS                                                         Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Corynebacterium xerosis                                                                         373     0.7                                                 Fusobacterium nucleatum                                                                         25586   1.3                                                 Haemophilum influenzae                                                                          19418   1.3                                                 Klebsiella pneumoniae                                                                           23357   1.8                                                 Legionella pneumophila                                                                          33152   0.0                                                 Mycoplasma pneumoniae                                                                           15531   3.0                                                 Neisseria meningitidis                                                                          13090   0.0                                                 Pseudomonas aeruginosa                                                                          25330   0.0                                                 Propionibacterium acnes                                                                         6919    1.1                                                 Streptococcus pneumoniae                                                                        6306    0.0                                                 Staphylococcus aureus                                                                           25923   1.5                                                 ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        HYBRIDIZATION OF THE M. AVIUM PROBE TO A                                      PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                               Organism           ATCC#   % Probe Bound                                      ______________________________________                                        Acinetobacter calcoaceticus                                                                      33604   0.0                                                Branhamella catarrahalis                                                                         25238   0.6                                                Bacillus subtilis  6051    0.9                                                Bacteroides fragiles                                                                             23745   1.0                                                Campylobacter jejuni                                                                             33560   0.4                                                Chromobacterium violaceum                                                                        29094   1.7                                                Clostridium perfringens                                                                          13124   2.1                                                Deinococcus radiodurans                                                                          35073   0.8                                                Derxia gummosa     15994   0.3                                                Enterobacter aerogenes                                                                           13048   0.6                                                Escherichia coli   11775   0.3                                                Mycobacterium gordonae                                                                           14470   1.9                                                Mycoplasma hominis 14027   3.3                                                Proteus mirabilis  29906   0.0                                                Psudomonas cepacia 11762   1.0                                                Rahnella aquatilis 33071   2.1                                                Rhodospirillum rubrum                                                                            11170   0.6                                                Streptococcus mitis                                                                              9811    0.9                                                Vibrio parahaemolyticus                                                                          17802   1.2                                                Yersinia enterocolitica                                                                          9610    0.4                                                ______________________________________                                    

EXAMPLE 2

After the alignment described in Example 1, the following sequence wascharacterized by the aforementioned criteria of length, Tm and sequenceanalysis and was determined to be specific for Mycobacteriumintracellulare:

    ACCGCAAAAGCTTTCCACCTAAAGACATGCGCCTAAAG

The sequence is complementary to a unique segment found in the 16S rRNAof Mycobacterium intracellulare. The size of the probe was 38 bases. Theprobe has a Tm of 75° C. and sequence analysis confirmed that the probewas correctly synthesized. The probe hybridizes to RNA of M.intracellulare in the region corresponding to bases 185-225 of E. coli16S rRNA.

To demonstrate the reactivity of this sequence for the Mycobacteriumintracellulare, the probe was tested in hybridization reactions underthe following conditions. ³² P-end-labelled oligonucleotide probe wasmixed with 1 microgram (7×10⁻¹³ moles) of purified rRNA fromMycobacterium intracellular and reacted in 0.12M PB (equimolar amountsof Na₂ HPO₄ and NaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodium dodecylsulfate) at 65° C. for 60 minutes in a final volume of 50 microliters.In separate tubes the probe was mixed with the hybridization buffer withand without target Mycobacterium intracellulare rRNA present. Followingseparation on hydroxyapatite as outlined previously the hybrids werequantitated by scintillation counting. These results are shown in Table5.

                  TABLE 5                                                         ______________________________________                                        HYBRIDIZATION OF THE M. INTRACELLULARE PROBE                                  TO HOMOLOGOUS TARGET rRNA*/                                                                     plus rRNA                                                                              minus rRNA                                         ______________________________________                                        M. intracellulare probe                                                                         85-95%   0.5%                                               ______________________________________                                         ##STR2##                                                                 

These data shows that the probe has a high extent of reaction to itshomologous target and very little non-specific binding to thehydroxyapatite.

Specificity of the Mycobacterium intracellulare probe was tested bymixing the ³² P labelled probe with rRNA released from cells from 29other species of mycobacteria by sonic disruption techniques describedin Murphy et. al. U.S. Pat. No. 5,374,522. All hybridization assays werecarried out as described in Example 1. Table 6 indicates that the probeis specific for Mycobacterium intracellulare and does not react with anyother mycobacterial species, including Mycobacterium avium. Theseresults are impressive in view of the 98% rRNA homology to M. avium; 98%homology to M. kansasii; 98% homology to M. asiaticum; and 97% homologyto M. tuberculosis.

                  TABLE 6                                                         ______________________________________                                        HYBRIDIZATION OF THE M. INTRACELLULARE PROBE                                  TO MYCOBACTERIAL SPECIES                                                      Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Mycobacterium africanum                                                                         25420   0.9                                                 M. asiaticum      25276   1.1                                                 M. avium          25291   1.3                                                 M. bovis          19210   1.1                                                 M. bovis (BCG)    19015   1.2                                                 M. chelonae       14472   1.0                                                 M. favescens      14474   1.2                                                 M. fortuitum      6841    1.3                                                 M. gastri         15754   1.3                                                 M. gordonae       14470   1.3                                                 M. haemophilum    29548   0.9                                                 M. intracellulare 13950   78.8                                                M. kansasii       12479   1.1                                                 M. malmoense      29571   1.0                                                 M. marinum        827     0.9                                                 M. nonchromogenicum                                                                             1930    1.0                                                 M. phlei          11758   1.1                                                 M. scrofulaceum   19981   1.0                                                 M. shimoidei      27962   1.3                                                 M. simiae         25275   1.1                                                 M. smegmatis      e14468  1.3                                                 M. szulgai        23069   1.0                                                 M. terrae         15755   1.4                                                 M. thermoresistibile                                                                            19527   1.6                                                 M. triviale       23292   1.3                                                 M. tuberculosis (avirulent)                                                                     25177   1.2                                                 M. tuberculosis (virulent)                                                                      27294   1.2                                                 M. ulcerans       19423   1.1                                                 M. vaccae         15483   1.0                                                 M. xenopi         19971   1.2                                                 ______________________________________                                    

As shown in Table 7 the probe did not react with the rRNA from any ofthe respiratory pathogens tested in the hybridization assay. Nor did theprobe react with any other closely related or phylogenetically morediverse species of bacteria that were tested (Table 8).

                  TABLE 7                                                         ______________________________________                                        HYBRIDIZATION OF THE M. INTRACELLULARE PROBE                                  TO RESPIRATORY PATHOGENS                                                      Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Corynebacterium xerosis                                                                         373     2.2                                                 Fusobacterium nucleatum                                                                         25586   1.5                                                 Haemophilum influenzae                                                                          19418   1.3                                                 Klebsiella pneumoniae                                                                           23357   1.2                                                 Legionella pneumophila                                                                          33152   1.2                                                 Mycoplasma pneumoniae                                                                           15531   3.2                                                 Neisseria meningitidis                                                                          13090   1.1                                                 Pseudomonas aeruginosa                                                                          25330   1.0                                                 Propionibacterium acnes                                                                         6919    2.9                                                 Streptococcus pneumoniae                                                                        6306    1.6                                                 Staphylococcus aureus                                                                           25923   1.3                                                 ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        HYBRIDIZATION OF THE M. INTRACELLULARE PROBE                                  TO A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                          Organism          ATTC#   % Probe                                             ______________________________________                                        Acinetobacter calcoaceticus                                                                     33604   1.5                                                 Branhamella catarrhalis                                                                         25238   1.8                                                 Bacillus subtilis 6051    1.7                                                 Bacteroides fragiles                                                                            23745   1.9                                                 Campylobacter jejuni                                                                            33560   1.9                                                 Chromobacterium violaceum                                                                       29094   1.4                                                 Clostridium perfringens                                                                         13124   2.1                                                 Deinococcus radiodurans                                                                         35073   2.1                                                 Derxia gummosa    15994   1.6                                                 Enterobacter aerogenes                                                                          13048   1.3                                                 Escherichia coil  11775   1.2                                                 Mycobacterium gordonae                                                                          14470   2.3                                                 Mycoplasna hominis                                                                              14027   2.6                                                 Proteus mirabilis 29906   1.2                                                 Pseudomonas cepacia                                                                             11762   1.7                                                 Rahnella aquatilis                                                                              33071   1.5                                                 Rbodospirillum rubrum                                                                           11170   1.4                                                 Strptococcus mitis                                                                              9811    1.4                                                 Vibrio parahaemolyticus                                                                         17802   2.5                                                 Yersinia enterocolitica                                                                         9610    1.1                                                 ______________________________________                                    

EXAMPLE 3

After the alignment described in Example 1, the following sequence wascharacterized by the aforementioned three criteria of size, sequence andTm, and was determined to be specific to the Mtb complex of organisms,Mycobacterium tuberculosis, Mycobacterium, africanum, Mycobacteriumbovis, and Mycobacterium microti:

    TAAAGCGCTTTCCACCACAAGACATGCATCCCGTG.                       1.

The sequence is complementary to a unique segment found in the 16S rRNAof the Mtb-complex bacteria. The size of the probe is 35 bases. Theprobe has a Tm of 72° C. and sequence analysis confirmed that the probewas correctly synthesized. It is capable of hybridizing in the regioncorresponding to bases 185-225 of E. coli 16S rRNA.

To demonstrate the reactivity of this sequence for the Mtb complex theprobe was tested in hybridization reactions under the followingconditions. ³² P-end-labelled oligonucleotide probe was mixed with 1microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacteriumtuberculosis and reacted in 0.12M PB hybridization buffer (equimolaramounts of Na₂ HPO₄, and NaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodiumdodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50microliters. In separate tubes the probe was mixed with thehybridization buffer with and without target rRNA from Mycobacteriumtuberculosis present. Following separation on hydroxyapatite as outlinedpreviously the hybrids were quantitated by scintillation counting. Theresults are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE                               TO HOMOLOGOUS TARGET rRNA*/                                                                   plus rRNA                                                                              minus rRNA                                           ______________________________________                                        Mtb complex probe                                                                             85-95%   0.5%                                                 ______________________________________                                         ##STR3##                                                                 

This data shows that the probe has a high extent of reaction tohomologous target and very little non-specific binding to thehydroxyapatite.

Specificity of the probe for the Mtb complex was tested by mixing the ³²p labelled probe with rRNA released from cells of the 4 Mtb complexbacilli and of 25 other mycobacterial species by sonic disruptiontechniques described in Murphy et. al., U.S. Pat. No. 5,374,522. Allhybridization assays were carried out as described in Example 1. Table10 indicates that the probe is specific for organisms within the Mtbcomplex and does not react with any other mycobacterial species.

                  TABLE 10                                                        ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE                               TO MYCOBACTERIAL SPECIES                                                      Organism          ATTC#   % Probe                                             ______________________________________                                        Mycobacterium africanum                                                                         25420   68.1                                                M. asiaticum      25276   3.4                                                 M. avium          25291   0.9                                                 M. bovis          19210   63.1                                                M. chelonae       14472   1.1                                                 M. flavescens     14474   0.9                                                 M. fortuitum      6841    1.1                                                 M. gastri         15754   0.8                                                 M. gordonae       14470   1.1                                                 M. haemophilum    29548   0.8                                                 M. intracallulare 13950   1.1                                                 M. kansasii       12479   1.3                                                 M. malmoense      29571   0.9                                                 M. marinum        827     1.1                                                 M. nonchromogenicum                                                                             1930    1.1                                                 M. phlei          11758   1.3                                                 M. scrofulaceum   19981   1.1                                                 M. shimoidei      27962   1.0                                                 M. simiae         25275   1.2                                                 M. smegmatis      e14468  0.9                                                 M. szulgai        23069   1.1                                                 M. terrae         15755   1.0                                                 M. thermoresistibile                                                                            19527   1.0                                                 M. triviale       23292   1.2                                                 M. tuberculosis (avirulent)                                                                     25177   66.2                                                M. tuberculosis (virulent)                                                                      27294   62.4                                                M. ulcerans       19423   0.9                                                 M. vaccae         15483   0.8                                                 M. xenopi         19971   2.6                                                 ______________________________________                                    

As shown in Table 11 the probe did not react with the rRNA from any ofthe respiratory pathogens tested in the hybridization assay. Nor did theprobe react with any other closely related or phylogenetically morediverse species of Bacteria that were tested (Table 12).

                  TABLE 11                                                        ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE                               TO RESPIRATORY PATHOGENS                                                      Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Corynebacterium xerosis                                                                         373     1.3                                                 Fusobacterium nucleatum                                                                         25586   1.0                                                 Haemophilum influenzae                                                                          19418   1.6                                                 Klebgiella pneumoniae                                                                           23357   1.2                                                 Legionella pneumophila                                                                          33152   1.4                                                 Mycoplasma pneumoniae                                                                           15531   1.1                                                 Neisseria meningitidis                                                                          13090   1.0                                                 Pseudomonas aeruginosa                                                                          25330   1.7                                                 Propionibacterium acnes                                                                         6919    1.2                                                 Streptococcus pneumoniae                                                                        25923   0.9                                                 ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        HYBRIDIZATION OF THE Mtb-COMPLEX 16S rRNA DNA PROBE                           TO A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                          Organism           ATCC#   % Probe                                            ______________________________________                                        Acinetobacter calcoaceticus                                                                      33604   1.3                                                Branhamella catarrhalis                                                                          25238   1.5                                                Bacillus subtilis  6051    1.3                                                Bacteroides fragilis                                                                             23745   1.3                                                Campylobacter jejuni                                                                             33560   1.1                                                Chromobacterium violaceum                                                                        29094   1.0                                                Clostridium perfringens                                                                          13124   1.2                                                Deinococcus radiodurans                                                                          35073   1.0                                                Derxia gummosa     15994   1.0                                                Enterobacter aerogenes                                                                           13048   1.0                                                Escherichia coli   11775   1.0                                                Mycobacterium gordonae                                                                           14470   1.3                                                Mycoplasma hominis 14027   0.5                                                Proteus mirabilis  29906   1.0                                                Pseudomonas cepacia                                                                              11762   2.6                                                Rahnella aquatilis 33071   1.9                                                Rhodospirillum rubrum                                                                            11170   1.0                                                Streptococcus mitis                                                                              9811    1.1                                                Vibrio parahaemolyticus                                                                          17802   0.9                                                Yersinia enterocolitica                                                                          9610    1.1                                                ______________________________________                                    

Two derivatives of the probe of Example 3 (numbered 2-3 below) were madeand tested:

    CCGCTAAAGCGCTTTCCACCACAAGACATGCATCCCG                      2.

    ACACCGCTAAAGCGCTTTCCACCACAAGACATGCATC.                     3.

All three probes have similar Tms (72° C.; 73.5° C.; and 72.3° C.,respectively) and similar hybridization characteristics.

Hybridization to Mycobacterium tuberculosis complex organisms was 68-75%and non-specific hybridization to hydroxyapatite was less than 2%.Results of hybridization assay tests for these derivatives follow.

                  TABLE 13                                                        ______________________________________                                        HYBRIDIZATION OF PROBE OF EXAMPLES 3 AND 2                                    DERIVATIVES THEREOF                                                           TO MYCOBACTERIAL SPECIES                                                                           Example                                                                       % Probe 1                                                                              % Probe 2                                                                             % Probe 3                               Organism    ATCC#    Bound    Bound   Bound                                   ______________________________________                                        Mycobacterium                                                                             25420    68.1     69.4    70.6                                    africanum                                                                     M. asiaticum                                                                              25274    3.4      5.3     1.8                                     M. avium    25291    0.9      1.6     1.4                                     M. bovis    19210    63.1     75.3    74                                      M. chelonae 14472    1.1      1.5     1.6                                     M. flavescans                                                                             14474    0.9      2.7     1.4                                     M. fortuitum                                                                              6841     1.1      3.6     1.5                                     M. gastri   15754    0.8      3.6     1.7                                     M. gordonae 14470    1.1      1.6     1.4                                     M. haemophilum                                                                            29548    0.8      3.2     1.7                                     M. intracellulare                                                                         13950    1.1      1.6     1.4                                     M. kansasii 12478    1.3      2.1     2.0                                     M. malmoense                                                                              29571    0.9      2.8     1.5                                     M. marinum  827      1.1      2.1     1.5                                     M. nonchromogenicum                                                                       1930     1.1      3.0     1.5                                     M. phlei    11758    1.3      1.3     1.1                                     M. scrofulaceum                                                                           19981    1.1      3.4     1.6                                     M. shimoidei                                                                              27962    1.0      2.7     1.6                                     M. simiae   25275    1.2      2.9     1.8                                     M. smegmatis                                                                              e14468   0.9      1.5     1.2                                     M. szulgai  23069    1.1      3.6     1.1                                     M. terrae   15755    1.0      3.7     2.0                                     M. thermoresistibile                                                                      19527    1.0      1.6     1.3                                     M. triviale 23292    1.2      1.6     2.0                                     M. tuberculosis                                                                           25177    66.2     75      68                                      (avirulent)                                                                   M. tuberculosis                                                                           27294    62.4     74      75                                      (virulent)                                                                    M. ulcerans 19423    0.9      1.7     3.0                                     M. vaccae   15483    0.8      1.4     1.2                                     M. xenopi   19971    2.6      1.4     1.2                                     ______________________________________                                    

EXAMPLE 4

The probe specific for the 23S rRNA of the M. tuberculosis complex wasobtained by using a primer which was complementary to a highly conservedregion of 23S rRNA. The sequence of this primer, derived from E. colirRNA, was 5'-AGG AAC CCT TGG GCT TTC GG-3'. Five nanograms of thisprimer was mixed with 1 microgram of rRNA from M. tuberculosis and otherclosely related Mycobacterium and the procedure as described forExamples 1, 2 and 3 was followed. After alignment as described inExample 1, the following sequence was determined to be specific to theMtb complex of organisms, Mycobacterium tuberculosis, Mycobacteriumafricanum, Mycobacterium bovis, and Mycobacterium microti:

    TGCCCTACCCACACCCACCACAAGGTGATGT.

The sequence is complementary to a unique segment found in the 23S rRNAof the Mtb-complex bacteria. The oligonucleotide probe was characterizedas previously described by the criteria of length, Tm and sequenceanalysis. The size of the probe is 31 bases. The probe has a Tm of 72.5°C. and sequence analysis confirmed that the probe was correctlysynthesized. It is capable of hybridizing in the region corresponding tobases 1155-1190 of E. coli 23S rRNA.

To demonstrate the reactivity of this sequence for the Mtb complex theprobe was tested in hybridization reactions under the followingconditions. ³² P-end-labelled oligonucleotide probes were mixed with 1microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacteriumtuberculosis and reacted in 0.12M PB hybridization buffer (equimolaramounts of Na₂ HPO₄, and NaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodiumdodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50microliters. In separate tubes the probe was mixed with thehybridization buffer with and without target rRNA from Mycobacteriumtuberculosis present. Following separation on hydroxyapatite as outlinedpreviously the hybrids were quantitated by scintillation counting. Theresults are shown in Table 14.

                  TABLE 14                                                        ______________________________________                                        HYBRIDIZATION OF THE Mtb-COMPLEX                                              23S rRNA DNA PROBE TO THE HOMOLOGOUS TARGET rRNA                                                plus rRNA                                                                              minus rRNA                                         ______________________________________                                        Mtb complex 23S probe                                                                           94%      1.2%                                               ______________________________________                                    

These data show that the probe has a high extent of reaction tohomologous target and very little non-specific binding to thehydroxyapatite.

Specificity of the probe for the Mtb complex was tested by mixing the ³²P labelled probe with rRNA released from cells of the four Mtb complexbacilli and of 25 other mycobacterial species by sonic disruptiontechniques described in Murphy et al., U.S. Pat. No. 5,374,522. Allhybridization assays were carried out as described in Example 1. Table14 indicates that the probe is specific for organisms within the Mtbcomplex and does not react with any other mycobacterial species.

                  TABLE 15                                                        ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 23S rRNA DNA PROBE                               TO MYCOBACTERIAL SPECIES                                                      Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Mycobacterium africanum                                                                         25420   33.6                                                M. asiaticum      25276   1.2                                                 M. avium          25291   1.0                                                 M. bovis          19210   32.0                                                M. chelonae       14472   1.2                                                 M. flavescans     14474   1.2                                                 M. fortuitum      6841    1.3                                                 M. gastri         15754   1.1                                                 M. gordonae       14470   1.2                                                 M. haemophilum    29548   1.2                                                 M. intracellulare 13950   1.1                                                 M. kansasii       12479   1.3                                                 M. malmoense      29571   1.3                                                 M. marinum        827     1.2                                                 M. nonchromogenicum                                                                             1930    1.0                                                 M. phlei          11758   1.0                                                 M. scrofulaceum   19981   1.1                                                 M. shimoidei      27962   1.2                                                 M. simiae         25275   1.3                                                 M. smegmatis      e14468  1.1                                                 M. szulgai        23069   1.1                                                 M. terrae         15755   1.0                                                 M. thermoresistibile                                                                            19527   1.2                                                 M. triviale       23292   1.0                                                 M. tuberculosis (avirulent)                                                                     25177   33.7                                                M. tuberculosis (virulent)                                                                      27294   38.1                                                M. ulcerans       19423   1.3                                                 M. vaccae         15483   1.0                                                 M. xenopi         19971   1.3                                                 ______________________________________                                    

EXAMPLE 5

Three additional Mycobacterium tuberculosis complex probes, Examples 5-7herein, were identified using two unique primers complementary to 23SrRNA. The first sequence is:

    CCATCACCACCCTCCTCCGGAGAGGAAAAGG.

The sequence of this Example 5 was obtained using a 23S primer with thesequence 5'-GGC CAT TAG ATC ACT CC-3'. It was characterized and shown tobe specific for the Mycobacterium tuberculosis complex of organismsincluding Mycobacterium tuberculosis, Mycobacterium africanum andMycobacterium bovis. This sequence, from 23S rRNA, is 31 bases in lengthand has a Tm of 72° C. This probe is capable of hybridizing to RNA ofthe aforementioned organisms in the region corresponding to bases540-575 of E. coli 23S rRNA.

To demonstrate the reactivity and specificity of this probe forMycobacterium tuberculosis complex, it was tested as a probe inhybridization reactions under the following conditions. ³² P-end-labeledoligonucleotide probe was mixed with rRNA released from cells of 30species of mycobacteria by the sonic disruption techniques described inMurphy et al., U.S. Pat. No. 5,374,522. 3×10⁷ cells were suspended in0.1 ml 5% SDS and sonicated for 15 minutes at 50°-60° C. One ml ofhybridization buffer (45% diisobutyl sulfosuccinate, 40 mM phosphatebuffer pH 6.8, 1 mM EDTA, 1 mM EGTA) was added and the mixture incubatedat 72° C. for 2 hours. Following incubation, 4 ml of 2% (w/v)hydroxyapatite, 0.12M sodium phosphate buffer pH 6.8, 0.02% SDS, 0.02%sodium azide was added and incubated at 72° C. for 5 minutes. The samplewas centrifuged and the supernatant removed. Four ml wash solution(0.12M sodium phosphate buffer pH6.8, 0.02% SDS, 0.02% sodium azide) wasadded and the sample was vortexed, centrifuged and the supernatantremoved. The radioactivity bound to the hydroxyapatite was determined byscintillation counting. The results are shown in Table 16 and indicatethat the probe is specific for the Mycobacterium tuberculosis complex oforganisms.

                  TABLE 16                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX                                  PROBE OF EXAMPLE 5 TO MYCOBACTERIAL SPECIES                                   Organism          ATCC #   % Probe Bound                                      ______________________________________                                        Mycobacterium africanum                                                                         25420    18.0                                               M. asiaticum      25274    2.6                                                M. avium          25291    3.4                                                M. bovis          19210    21.7                                               M. bovis (BCG)    35734    35.3                                               M. chelonae       14472    3.8                                                M. flavescans     14474    2.3                                                M. fortuitum      6841     1.8                                                M. gastri         15754    2.2                                                M. gordonae       14470    2.8                                                M. haemophilum    29548    2.8                                                M. intracellulare 13950    2.1                                                M. kansasii       12478    1.6                                                M. malmoense      29571    2.3                                                M. marinum        827      2.1                                                M. nonchromogenicum                                                                             1930     2.3                                                M. phlei          11758    2.1                                                M. scrofulaceum   19981    2.2                                                M. shimoidei      27962    1.9                                                M. simiae         25275    2.2                                                M. smegmatis      e14468   2.0                                                M. szulgai        23069    2.2                                                M. terrae         15755    2.2                                                M. thermoresistibile                                                                            19527    2.2                                                M. triviale       23292    2.0                                                M. tuberculosis (avirulent)                                                                     25177    26.4                                               M. tuberculosis (virulent)                                                                      27294    36.6                                               M. ulcerans       19423    2.5                                                M. vaccae         15483    2.4                                                M. xenopi         19971    2.8                                                ______________________________________                                    

Table 16 shows that the probe also did not cross react with RNA from anyof the closely related organisms tested by the method just described.

                  TABLE 17                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX PROBE                            OF EXAMPLE 5 TO PHYLOGENETICALLY                                              CLOSELY RELATED ORGANISMS                                                     Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Actinomadura madurae                                                                            19425   2.1                                                 Actinoplanes italicus                                                                           10049   3.1                                                 Arthrobacter oxidans                                                                            14358   2.1                                                 Brevibacterium linens                                                                           e9172   1.9                                                 Corynebacterium xerosis                                                                         373     2.2                                                 Dermatophilus congolensis                                                                       14367   2.2                                                 Microbacterium lacticum                                                                         8180    2.1                                                 Nocardia asteroides                                                                             19247   2.0                                                 Nocardia brasiliensis                                                                           19296   2.2                                                 Nocardia otitidis-caviarum                                                                      14629   2.0                                                 Nocardioposis dassonvillei                                                                      23218   4.0                                                 Oerskovia turbata 33225   2.2                                                 Oerskovia xanthineolytica                                                                       27402   2.0                                                 Rhodococcus aichiensis                                                                          33611   1.9                                                 Rhodococcus aurantiacus                                                                         25938   2.0                                                 Rhodococcus bronchialis                                                                         25592   2.1                                                 Rhodocaccus chubuensis                                                                          33609   2.3                                                 Rhodococcus equi  6939    2.4                                                 Rhodococcus obuensis                                                                            33610   2.2                                                 Rhodococcus sputi 29627   2.3                                                 ______________________________________                                    

EXAMPLE 6

The second Mycobacterium tuberculosis complex probe was obtained using a23S primer with the sequence 5' CCT GAT TGC CGT CCA GGT TGA GGG AAC CTTTGG G-3'. Its sequence is:

    CTGTCCCTAAACCCGATTCAGGGTTCGAGGTTAGATGC

This sequence, from 23S rRNA, is 38 bases in length and has a Tm of 75°C. It hybridizes in the region corresponding to bases 2195-2235 of E.coli 23S rRNA.

Like the complex probe in Example 5, this sequence was characterized andshown to be specific for the Mycobacterium tuberculosis complex oforganisms including Mycobacterium tuberculosis, Mycobacterium africanumand Mycobacterium bovis.

To demonstrate the reactivity and specificity of the probe of thisExample 6 to Mycobacterium tuberculosis complex, it was tested as aprobe in hybridization reactions under the conditions described for theprobe in Example 5. The results are shown in Table 18 and indicate thatthe probe is specific for the Mycobacterium tuberculosis complex oforganisms with the exception of Mycobacterium thermoresistibile, a rareisolate which is not a human pathogen.

                  TABLE 18                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX                                  PROBE OF EXAMPLE 6 TO MYCOBACTERIAL SPECIES                                   Organism          ATCC #   % Probe Bound                                      ______________________________________                                        Mycobacterium africanum                                                                         25420    56.0                                               M. asiaticum      25274    3.1                                                M. avium          25291    2.6                                                M. bovis          19210    48.0                                               M. bovis (BCG)    35734    63.0                                               M. chelonae       14472    2.8                                                M. flavescans     14474    2.8                                                M. fortuitum      6841     3.0                                                M. gastri         15754    3.2                                                M. gordonae       14470    3.0                                                M. haemophilum    29548    3.0                                                M. intracellulare 13950    3.6                                                M. kansasii       12478    3.9                                                M. malmoense      29571    2.9                                                M. marinum        827      2.9                                                M. nonchromogenicum                                                                             1930     4.8                                                M. phlei          11758    2.9                                                M. scrofulaceum   19981    2.6                                                M. shimoidei      27962    3.6                                                M. simiae         25275    3.3                                                M. smegmatis      e14468   3.0                                                M. szulgai        23069    2.8                                                M. terrae         15755    2.8                                                M. thermoresistibile                                                                            19527    11.7                                               M. triviale       23292    3.2                                                M. tuberculosis (avirulent)                                                                     25177    65.0                                               M. tuberculosis (virulent)                                                                      27294    53.0                                               M. ulcerans       19423    2.5                                                M. vaccae         15483    2.8                                                M. xenopi         19971    3.3                                                ______________________________________                                    

Table 19 shows that the probe also did not cross react with RNA from anyof the phylogenetically closely related organisms tested by the methodjust described.

                  TABLE 19                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX PROBE                            OF EXAMPLE 6 TO PHYLOGENETICALLY                                              CLOSELY RELATED ORGANISMS                                                     Organism          ATCC #   % Probe Bound                                      ______________________________________                                        Actinomadura madurae                                                                            19425    1.3                                                Actinoplanes italicus                                                                           10049    0.6                                                Arthrobacter oxidans                                                                            14358    1.1                                                Brevibacterium linens                                                                           e9172    0.8                                                Corynebacterium xerosis                                                                         373      1.0                                                Dermatophilus congolensis                                                                       14367    0.6                                                Microbacterium lacticum                                                                         8180     1.9                                                Nocardia asteroides                                                                             19247    0.9                                                Nocardia brasiliensis                                                                           19296    0.8                                                Nocardia otitidis-caviarum                                                                      14629    1.5                                                Nocardioposis dassonvillei                                                                      23218    0.5                                                Oerskovia turbata 33225    0.3                                                Oerskovia xanthineolytica                                                                       27402    0.8                                                Rhodococcus aichiensis                                                                          33611    1.6                                                Rhodococcus aurantiacus                                                                         25938    0.7                                                Rhodococcus bronchialis                                                                         25592    1.5                                                Rhodocaccus chubuensis                                                                          33609    0.8                                                Rhodococcus equi  6939     0.3                                                Rhodococcus obuensis                                                                            33610    0.8                                                Rhodococcus sputi 29627    1.4                                                ______________________________________                                    

EXAMPLE 7

The following additional Mycobacterium tuberculosis complex probe alsohas been identified using a 23S primer with the same sequence as that ofExample 6, namely, 5'-OCT GAT TGC CGT CCA GGT TGA GGG AAC CTT TGG G-3':

    AGGCACTGTCCCTAAACCCGATTCAGGGTTC.

This sequence, from 23S rRNA is 31 bases in length and has a Tm of 71°C. It hybridizes in the region corresponding to bases 2195-2235 of E.coli 23S rRNA. As is the case with the Mycobacterium tuberculosiscomplex probes of Examples 5 and 6 herein, this sequence also wascharacterized and shown to be specific for the Mycobacteriumtuberculosis complex of organisms, including Mycobacterium tuberculosis,Mycobacterium africanum and Mycobacterium bovis.

To demonstrate the reactivity and specificity of this probe forMycobacterium tuberculosis complex, it was tested as a probe inhybridization reactions under the conditions described for the probe ofExample 5. Table 20 shows that the probe is specific for theMycobacterium tuberculosis complex of organisms.

                  TABLE 20                                                        ______________________________________                                        HYBRIDIZATION OF THE MYCOBACTERIUM TUBERCULOSIS                               COMPLEX PROBE OF EXAMPLE 7 TO MYCOBACTERIAL SPECIES                           Organism          ATCC #   % Probe Bound                                      ______________________________________                                        Mycobacterium africanum                                                                         25420    43.0                                               M. asiaticum      25274    0.6                                                M. avium          25291    0.7                                                M. bovis          19210    43.0                                               M. bovis (BCG)    35734    46.0                                               M. chelonae       14472    0.6                                                M. flavescans     14474    0.6                                                M. fortuitum      6841     0.5                                                M. gastri         15754    0.9                                                M. gordonae       14470    0.7                                                M. haemophilum    29548    0.6                                                M. intracellulare 13950    0.6                                                M. kansasii       12478    0.9                                                M. malmoense      29571    0.8                                                M. marinum        827      0.7                                                M. nonchromogenicum                                                                             1930     0.8                                                M. phlei          11758    0.6                                                M. scrofulaceum   19981    0.7                                                M. shimoidei      27962    0.8                                                M. simiae         25275    0.7                                                M. smegmatis      e14468   0.6                                                M. szulgai        23069    0.6                                                M. terrae         15755    0.7                                                M. thermoresistibile                                                                            19527    0.9                                                M. triviale       23292    0.7                                                M. tuberculosis (avirulent)                                                                     25177    40.0                                               M. tuberculosis (virulent)                                                                      27294    50.0                                               M. ulcerans       19423    0.7                                                M. vaccae         15483    0.4                                                M. xenopi         19971    0.6                                                ______________________________________                                    

Table 21 shows that the probe also did not cross react with RNA from anyof the closely related organisms tested by the method just described.

                  TABLE 21                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX                                  PROBE OF EXAMPLE 7 TO PHYLOGENETICALLY                                        CLOSELY RELATED ORGANISMS                                                     Organism          ATCC #   % Probe Bound                                      ______________________________________                                        Actinomadura madurae                                                                            19425    1.0                                                Actinoplanes italicus                                                                           10049    0.6                                                Arthrobacter oxidans                                                                            14358    0.4                                                Brevibacterium linens                                                                           e9172    0.8                                                Corynebacterium xerosis                                                                         373      0.6                                                Dermatophilus congolensis                                                                       14367    0.8                                                Microbacterium lacticum                                                                         8180     0.5                                                Nocardia asteroides                                                                             19247    0.7                                                Nocardia brasiliensis                                                                           19296    0.5                                                Nocardia otitidis-caviarum                                                                      14629    0.6                                                Nocardioposis dassonvillei                                                                      23218    0.6                                                Oerskovia turbata 33225    0.8                                                Oerskovia xanthineolytica                                                                       27402    0.6                                                Rhodococcus aichiensis                                                                          33611    0.7                                                Rhodococcus aurantiacus                                                                         25938    0.7                                                Rhodococcus bronchialis                                                                         25592    0.6                                                Rhodocaccus chubuensis                                                                          33609    0.6                                                Rhodococcus equi  6939     0.6                                                Rhodococcus obuensis                                                                            33610    0.6                                                Rhodococcus sputi 29627    0.9                                                ______________________________________                                    

Notably, overlapping probes may have identical specificity. Compare, forexample, the probes of Examples 6 and 7:

    CTGTCCCTAAACCCGATTCAGGGTTCGAGGTTAGATGC                     Ex. 6

    AGGCACTGTCCCTAAACCCGATTCAGGGTTC                            Ex. 7

There may be several sequences from a particular region which will yieldprobes with the desired hybridization characteristics. In other cases,one probe sequence may be significantly better than another probediffering by a single base. In general, the greater the sequencedifference (% mismatch) between a target and nontarget organism, themore likely one will be able to alter the probe without affecting itsusefulness for a specific application. This phenomenon also wasdemonstrated by the derivative probes in Example 3.

In Example 7, five bases were added to the 5' end of the probe inExample 6, and 12 bases were removed from the 3' end. The two probeshave essentially identical hybridization characteristics.

EXAMPLE 8

The Mycobacterium genus is particularly difficult to distinguish fromNorcardia, Corvnebacterium and Rhodococcus. These genera have commonantigens, precipitins and G & C counts. Despite the fact that theseorganisms also exhibit 92-94% rRNA homology to the above listedMycobacterium organisms, we have designed probes which detect allmembers of the genus Mycobacterium without cross reacting to the relatedgenera.

In addition to the Mycobacterium species probes already disclosed, fourprobes specific for members of the Mycobacterium genus were identifiedusing one primer complementary to 16S rRNA and one primer complementaryto 23S rRNA. Sequence 1 was obtained using a 16S primer with thesequence 5'-TTA CTA GCG ATT CCG ACT TCA-3'. Sequences 2, 3 and 4 wereobtained using a 23S primer with the sequence 5'-GTG TCG GTT TTG GGTACG-3'. Sequence 1 is capable of hybridizing to RNA of the genusMycobacterium in the region corresponding to bases 1025-1060 of E. coli16S rRNA. Sequences 2-4 hybridize in regions corresponding to thefollowing bases of E. coli 23S rRNA in our numbering system (See FIG.2); 1440-1475; 1515-1555; 1570-1610 in our numbering system.

The following sequences were characterized and shown to be specific forthe genus Mycobacterium:

1. CCA TGC ACC ACC TGC ACA CAG GCC ACA AGG

2. GGC TTG CCC CAG TAT TAC CAC TGA CTG GTA CGG

3. CAC CGA ATT CGC CTC AAC CGG CTA TGC GTC ACC TC

4. GGG GTA CGG CCC GTG TGT GTG CTC GCT AGA GGC

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 73° C.Sequence 2, from 23S rRNA, is 33 bases in length and has a Tm of 75° C.Sequence 3, from 23S rRNA, is 35 bases in length and has a Tm of 76° C.Sequence 4, from 23S rRNA, is 33 bases in length and has a Tm of 73° C.

To demonstrate the reactivity and specificity of probe 1 for members ofthe genus Hycobacterium, it was tested as a probe in hybridizationreactions under the following conditions. ¹²⁵ I-labeled oligonucleotideprobe was mixed with rRNA released from cells of 30 species ofmycobacteria by the sonic disruption techniques described in Murphy etal., U.S. Pat. No. 5,374,522. 3×10⁷ cells were suspended in 0.1 ml 5%SOS and sonicated for 15 minutes at 50°-60° C. One ml of hybridizationbuffer (45% diisobutyl sulfosuccinate, 40 mM sodium phosphate pH 6.8, 1mM EDTA, 1 mM EGTA) was added and the mixture incubated at 72° C. for 2hours. Following incubation, 2 ml of separation solution (containing 2.5g/1 cationic magnetic microspheres, 0.17M sodium phosphate buffer pH6.8, 7.5% Triton X-100 (TM), 0.02% sodium azide) was added and incubatedat 72° C. for 5 minutes. The RNA:probe hybrids, bound to the magneticparticles, were collected and the supernatant removed. One ml washsolution (0.12M sodium phosphate buffer pH 68, 14% diisobutylsulfosuccinate, 5% Triton X-100, 0.02% sodium azide) has added, theparticles collected and the supernatant removed, This step was repeatedtwo times. The radioactivity bound to the magnetic particles wasdetermined in a gamma counter. The results are shown in Table 22 andindicate that the probes hybridize to organisms in the genusMycobacterium and that a combination of probes will detect all membersof the genus. Table 23 shows that the probes do not react with otherclosely related bacteria.

                  TABLE 22                                                        ______________________________________                                        HYBRIDIZATION OF THE MYCOBACTERIUM                                            PROBES 1-4 TO MYCOBACTERIAL SPECIES                                                              %       %      %     %                                                        Probe   Probe  Probe Probe                                                    1       2      3     4                                     Organism  ATCC#    Bound   Bound  Bound Bound                                 ______________________________________                                        Mycobacterium                                                                           25420    41.5    14.7   17.9  26.7                                  africanum                                                                     M. asiaticum                                                                            25274    31.8    20.2   7.9   0.1                                   M. avium  25291    11.7    34.7   10.1  1.6                                   M. bovis  19210    19.4    28.4   44.6  20.9                                  M. bovis (BCG)                                                                          35734    30.0    35.5   17.8  5.6                                   M. chelonae                                                                             14472    8.6     0.7    6.3   0.2                                   M. flavescens                                                                           14474    29.8    17.7   2.3   0.9                                   M. fortuitum                                                                            6841     34.7    2.2    4.8   0.2                                   M. gastri 15754    27.6    65.1   9.6   22.3                                  M. gordonae                                                                             14470    50.7    55.2   3.1   0.4                                   M. haemophilum                                                                          29548    40.7    60.7   0.4   12.4                                  M. intracellulare                                                                       13950    38.8    48.3   0.9   5.4                                   M. kansasii                                                                             12478    53.4    27.3   24.5  27.8                                  M. malmoense                                                                            29571    3.1     38.4   0.8   1.5                                   M. marinum                                                                              827      41.7    4.1    4.8   0.1                                   M. non-   1930     35.0    42.9   0.5   16.4                                  chromogenicum                                                                 M. phlei  11758    23.7    0.6    1.8   0.6                                   M. scrofulaceum                                                                         19981    35.1    66.9   0.9   26.4                                  M. shimoidei                                                                            27962    34.6    1.4    1.3   4.8                                   M. simiae 25275    45.9    44.0   5.3   0.1                                   M. smegmatis                                                                            e14468   31.3    4.0    5.6   0.1                                   M. szulgai                                                                              23069    19.4    22.3   1.5   3.0                                   M. terrae 15755    25.6    21.7   0.4   12.3                                  M. thermo-                                                                              19527    20.3    34.5   3.1   17.6                                  resistibile                                                                   M. triviale                                                                             23292    37.3    4.6    4.3   0.1                                   M. tuberculosis                                                                         25177    38.5    26.3   11.3  23.0                                  (avirulent)                                                                   M. tuberculosis                                                                         27294    13.8    12.4   38.4  22.3                                  (virulent)                                                                    M. ulcerans                                                                             19423    33.9    28.7   0.4   8.9                                   M. vaccae 15483    8.8     36.2   4.8   3.2                                   M. xenopi 19971    38.4    2.1    3.8   0.2                                   ______________________________________                                    

                  TABLE 23                                                        ______________________________________                                        HYBRIDIZATION OF THE MYCOBACTERIUM PROBES                                     1-4 TO PHYLOGENETICALLY CLOSELY RELATED ORGANISMS                                              % Probe  % Probe                                                                              % Probe                                                                              % Probe                               Organism ATCC#   1 Bound  2 Bound                                                                              3 Bound                                                                              4 Bound                               ______________________________________                                        Actinomadura                                                                           19425   0.2      0.3    0.2    0.1                                   madurae                                                                       Actinoplanes                                                                           10049   0.4      0.5    0.3    0.2                                   italicus                                                                      Arthrobacter                                                                           14358   0.2      0.4    0.3    0.1                                   oxidans                                                                       Brevibacterium                                                                         e9l72   0.3      0.3    0.3    0.1                                   linens                                                                        Corynebacter-                                                                          373     0.4      0.3    0.3    0.1                                   ium xerosis                                                                   Dermatophilus                                                                          14367   0.4      0.6    0.3    0.2                                   congolensis                                                                   Microbacter-                                                                           8180    0.2      0.3    0.2    0.1                                   ium lacticum                                                                  Nocardia 19247   0.3      0.3    0.4    0.1                                   asteroides                                                                    Nocardia 19296   0.4      0.3    0.6    0.1                                   brasiliensis                                                                  Nocardia 14629   0.4      0.4    1.0    0.3                                   otitidis-                                                                     caviarum                                                                      Nocardioposis                                                                          23218   0.3      0.2    0.3    0.1                                   dassonvillei                                                                  Oerskovia                                                                              33225   0.2      0.2    0.3    0.1                                   turbata                                                                       Oerskovia                                                                              27402   0.2      0.3    0.3    0.1                                   xanthineolytica                                                               Rhodococcus                                                                            33611   0.4      0.2    0.3    0.2                                   aichiensis                                                                    Rhodococcus                                                                            25938   0.3      0.4    0.3    0.2                                   aurantiacus                                                                   Rhodococcus                                                                            25592   0.4      0.3    0.3    0.1                                   bronchialis                                                                   Rhodococcus                                                                            33609   0.6      0.4    0.3    0.3                                   chubuensis                                                                    Rhodococcus                                                                            6939    0.4      0.4    0.4    0.5                                   equi                                                                          Rhodococcus                                                                            33610   0.5      0.5    0.3    0.1                                   obuensis                                                                      Rhodococcus                                                                            29627   0.4      0.5    0.4    0.3                                   sputi                                                                         ______________________________________                                    

EXAMPLE 9

Mycoplasmas are small, aerobic bacteria lacking cell walls. Mycoplasmapneumoniae is estimated to cause 8-15 million infections per year. Theinfections may be asymptomatic or range in severity from mild to severebronchitis and pneumonia. The organism is believed to cause about 10% ofpneumonias in the general population and 10-50% of the pneumonias ofmembers of groups in prolonged, close contact such as college studentsand military personnel.

Diagnosis until now has required isolation of the organism in culture ordemonstration of an increase in antibody titer. Culturing of theorganism involves inoculation of respiratory tract specimens onto agaror biphasic media containing bacterial growth inhibitors. Examinationfor growth at 3-4 and 7-10 days is used to establish the presence orabsence of any mycoplasma. Mycoplasma pneumoniae must then be identifiedby hemadsorption (the ability of M. pneumoniae to adhere sheep or guineapig erythrocytes), hemolysis (the ability of M. pneumoniae to producebeta hemolysis of sheep or guinea pig erythrocytes in blood agar),growth inhibition by specific antibodies, or immunofluorescence Withspecific antibodies. The present invention has significant advantagesover each of these prior art methods both because of the simplicity ofthe test and because of the greatly reduced time necessary to achieve adiagnosis.

A probe specific for the 5S rRNA of M. pneumoniae was obtained by acomparison of known rRNA sequences. The particular sequences alignedwere from M. pneumoniae, M. gallisepticum and Ureaplasma urealyticum(Rogers, M. J. et al. 1985, Proc. Natl. Acad. Sci. USA, 82 (1160-1164),M. capricolum (Hori, H. et al. 1981, Nucl. Acids Res. 9, 5407-5410) andSpiroplasma sp. (Walker, R. T. et al. 1982 Nucl. Acids Res. 10,6363-6367). The alignments were performed as described above andoutlined at page 6. 5S rRNA can be isolated and sequenced as outlined inRogers et al., or a primer can be made which is complementary to aconserved region in the 5S rRNA and sequencing performed as outlined inExamples 1-4. The conserved region of 5S rRNA is documented in Fox, G.E. and Woese, C. R., 1975, Nature 256: 505-507. The following sequencewas determined to be specific for Mycoplasma pneumoniae:

    GCTTGGTGCTTTCCTATTCTCACTGAAACAGCTACATTCGGC.

The sequence is complementary to a unique segment found in the 5S rRNAof Mycoplasma pneumoniae in the region corresponding to bases 65-108 ofE. coli 5S rRNA, and was selected by comparison to 5S rRNA sequencesfrom Mycoplasma gallisepticum, Spiroplasma mirum and Ureaplasmaurealyticum. The oligonucleotide probe was characterized as describedabove. The size of the probe was 42 bases. The probe has a Tm of 71.5°C.

To demonstrate the reactivity of this sequence for Mycoplasmapneumoniae, the probe was tested in hybridization reactions under thefollowing conditions. ³² P-end-labelled oligonucleotide probe was mixedwith 1 microgram (7×10⁻¹³ moles) of purified rRNA from Mycoplasmapneumoniae and reacted in 0.12M PB (equimolar amounts of Na₂ HPO₄ andNaH₂ PO₄), 1 mM EDTA and 0.02% SDS (sodium dodecyl sulfate) at 65° C.for 60 minutes in a final volume of 50 microliters. In separate tubesthe probe was mixed with the hybridization buffer with and withouttarget Mycoplasma pneumoniae rRNA present. Following separation onhydroxyapatite as outlined previously the hybrids were quantitated byscintillation counting. These results are shown in Table 24.

                  TABLE 24                                                        ______________________________________                                        HYBRIDIZATION OF THE M. PNEUMONIAE 5S rRNA DNA                                PROBE TO HOMOLOGOUS TARGET rRNA*/                                                               plus rRNA                                                                              minus rRNA                                         ______________________________________                                        M. pneumoniae 5S probe                                                                          85-95%   0.5%                                               ______________________________________                                         ##STR4##                                                                     -  This data shows that the probe has a high extent of reaction to its         homologous target and very little non-specific binding to the                  hydroxyapatite.                                                           

Specificity of the M. pneumoniae 5S probe was tested by mixing the ³² Plabelled probe with rRNA released from cells from other Mycoplasmaspecies. All hybridization assays were carried out as described inExample 1. Table 25 indicates that the probe is specific for Mycoplasmapneumoniae and does not react with any other Mycoplasma species.

                  TABLE 25                                                        ______________________________________                                        HYBRIDIZATION OF M. PNEUMONIAE PROBE TO                                       OTHER MYCOPLASMA SPECIES                                                      ______________________________________                                        Acholeplasma laidlawii                                                                           14089   3.3                                                M. buccale         23636   1.7                                                M. capricolum      23205   2.4                                                M. columbinsale    33549   1.4                                                M. faucium         25293   1.4                                                M. fermentans      15474   1.0                                                M. gallisepticum   19610   1.8                                                M. gallopavonis    33551   1.6                                                M. genitalium      3353c   1.7                                                M. hominis         14027   1.3                                                M. orale           23714   1.8                                                M. pneumoniae      15531   78.0                                               M. primatum        15497   1.6                                                M. salivarium      23064   0.6                                                Spiroplasma mirum          2.3                                                ______________________________________                                    

As shown in Table 26, the probe did not react with any other closelyrelated or phylogenetically diverse species of bacteria.

                  TABLE 26                                                        ______________________________________                                        HYBRIDIZATION OF M. PNEUMONIAE PROBE TO                                       A PHYLOGENETIC CROSS SECTION OF BACTERIA                                      Organism            ATCC#   % Probe Bound                                     ______________________________________                                        Corynebacterium xerosis                                                                           373     1.4                                               Haemophilus influenzae                                                                            19418   1.4                                               Klebsiella pneumoniae                                                                             23357   1.3                                               Legionella pneumophila                                                                            33152   1.8                                               Mycobacterium tuberculosis (avir)                                                                 25177   1.6                                               Mycoplasma pneumoniae                                                                             15531   52                                                Neisseria meningitidis                                                                            13077   0.6                                               Propionibacterium acnes                                                                           6919    2.0                                               Pseudomonas aeruginosa                                                                            25330   1.6                                               Staphylococcus aureus                                                                             12598   2.0                                               Streptococcus pneumoniae                                                                          c6306   1.9                                               ______________________________________                                    

Four additional probe sequences (numbered 2-5 below) specific forMycoplasma pneumoniae were obtained by utilizing four unique primerscomplementary to conserved regions on 16S rRNA. The regions correspond,respectively, to bases 190-230; 450-490; 820-860; and 1255-1290 of E.coli 16S rRNA. Probe sequence #1 was obtained using a primer with thesequence 5'-GGCCGTTACCCCACCTACTAGCTAAT-3'. Probe sequence #2 wasobtained with a primer with the sequence 5'-GTATTACCGCGGCTGCTGGC-3'.Probe sequence #3 was obtained with a primer with the sequence5'-CCGCTTGTGCGGGCCCCCGTCAATTC-3'. Probe sequence #4 was obtained using aprimer with the sequence 5'-CGATTACTAGCGATTCC-3'. Sequencing reactionswere performed as outlined in previous examples. The M. pneumoniaesequences were compared with sequences from Mycoplasma genitalium,Mycoplasma capricolum, Mycoplasma gallisepticum and Spiroplasma mirum.

The following probe sequences were characterized by criteria describedin Example 1 of the parent application and were shown to be specific forMycoplasma pneumoniae:

2. AATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT

3. CAGTCAAACTCTAGCCATTACCTGCTAAAGTCATT

4. TACCGAGGGGATCGCCCCGACAGCTAGTAT

5. CTTTACAGATTTGCTCACTTTTACAAGCTGGCGAC.

Probe #2 is 35 bases in length and has a Tm of 67° C. Probe #3 is 35bases in length and has a Tm of 66° C. Probe #4 is 30 bases in lengthand has a Tm of 69° C. Probe #5 is 35 bases long with a Tm of 66° C.

When the four probes were mixed and used in hybridization assays at 60°C. in the same manner as previous examples, they were found to bespecific for M. pneumoniae. The probes do not cross react with otherrespiratory pathogens or with any organism representing the bacterialphylogenetic tree (Table 28).

                  TABLE 27                                                        ______________________________________                                        HYBRIDIZATION OF MYCOPLASMA PNEUMONIAE                                        PROBES 2-5 TO MYCOPLASMA SPECIES                                              Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Acholeplasma axanthum                                                                           27378   0.34                                                Acholeplasma laidlawii                                                                          14089   0.30                                                Mycoplasma arginini                                                                             23838   0.20                                                Mycoplasma arthritidis                                                                          19611   0.49                                                Mycoplasma bovigenitalium                                                                       19852   0.18                                                Mycoplasma bovis  25523   0.43                                                Mycoplasma buccale                                                                              23636   0.37                                                Mycoplasma californicum                                                                         33451   0.79                                                Mycoplasma capricolum                                                                           23205   0.38                                                Mycoplasma columbinasale                                                                        33549   0.54                                                Mycoplasma columborale                                                                          29258   0.50                                                Mycoplasma faucium                                                                              25293   0.45                                                Mycoplasma fermentans                                                                           15474   0.27                                                Mycoplasma gallisepticum                                                                        19610   0.25                                                Mycoplasma gallopavonis                                                                         33551   0.47                                                Mycoplasma genitalium                                                                           33530   2.5                                                 Mycoplasma hominis                                                                              14027   0.52                                                Mycoplasma hyorhinis                                                                            17981   0.46                                                Mycoplasma orale  23714   0.56                                                Mycoplasma pneumoniae                                                                           15531   34.0                                                Mycoplasma primatum                                                                             15497   0.71                                                Mycoplasma pulmonis                                                                             19612   0.68                                                Mycoplasma salivarium                                                                           23064   0.46                                                Spiroplasma citri 29416   0.60                                                Spiroplasma mirum 29335   0.52                                                ______________________________________                                    

                  TABLE 28                                                        ______________________________________                                        HYBRIDIZATION OF MYCOPLASMA PNEUMONIAE                                        PROBES 2-5 WITH OTHER BACTERIA                                                Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Actinomyces israelii                                                                            10049   1.0                                                 Bacteroides fragilis                                                                            23745   1.4                                                 Bifidobacterium breve                                                                           15700   1.0                                                 Bordetella bronchiseptica                                                                       10580   0.9                                                 Clostridium innocuum                                                                            14501   1.0                                                 Clostridium pasteurianum                                                                        6013    0.9                                                 Clostridium perfringens                                                                         13124   1.1                                                 Clostridium ramosum                                                                             25582   1.0                                                 Corynebacterium xerosis                                                                         373     0.8                                                 Erysipelothrix rhusiopathiae                                                                    19414   1.1                                                 Escherichia coli  11775   1.0                                                 Haemophilus influenzae                                                                          19418   0.9                                                 Klebsiella pneumoniae                                                                           15531   1.0                                                 Lactobacillus acidophilus                                                                       4356    1.4                                                 Legionella pneumophila                                                                          33154   0.8                                                 Listeria monocytogenes                                                                          15313   1.2                                                 Moraxella osloensis                                                                             19976   1.1                                                 Mycobacterium tuberculosis                                                                      25177   1.0                                                 Neisseria meningitidis                                                                          13077   1.0                                                 Pasteurella multocida                                                                           6529    1.6                                                 Peptococcus magnus                                                                              14955   0.9                                                 Propionibacterium acnes                                                                         6919    1.1                                                 Pseudomonas aeruginosa                                                                          25330   1.0                                                 Staphylococcus aureus                                                                           12600   1.0                                                 Streptococcus faecalis                                                                          19433   1.5                                                 Streptococcus mitis                                                                             9811    1.0                                                 Streptococcus pneumoniae                                                                        6306    1.0                                                 Streptococcus pyogenes                                                                          19615   1.1                                                 ______________________________________                                    

EXAMPLE 10

The genus Legionella contains 22 species which are all potentiallypathogenic for humans. These organisms cause Legionnaires' disease, anacute pneumonia, or Pontiac fever, an acute, non-pneumonic, febrileillness that is not fatal.

Legionella species have also been shown to be responsible for nosocomielpneumonia occuring predominantly among immunocompromised patients.

Legionellosis, which includes Legionneires' disease and Pontiac fever,is diagnosed on the basis of clinical symptoms, either direct orindirect fluorescence antibody tests, and by culture using a bufferedcharcoal yeast extract (BCYE) agar containing selective antimicrobialagents. There is no single definitive genus test known in the prior art.(See Bergey's Manual of Systematic Bacteriology at page 283, (ed.1984)). The fluorescent antibody tests are not able to identify allspecies of Legionella, but only those few for which antibodies exist.The culture method is not definitively diagnostic for Legionellaspecies.

The oligonucleotide sequences described below, when used as probes in anucleic acid hybridization assay, accurately identify all species ofLegionella. This assay is more sensitive than culture or antibody testsand shortens significantly the time of identification and, thus,diagnosis. The assay, therefore, represents a significant improvementover prior diagnostic methods.

Three probe sequences specific for the genus Legionella were obtained byutilizing three unique primers complementary to conserved regions onboth 16S and 23S rRNA. Sequence 1 was obtained by using a 16S primerwith the sequence 5'-TCT ACG CAT TTC ACC GCT ACA C-3'. Probe sequence 2was obtained with a 23S primer of sequence 5'-CAG TCA GGA GTA TTT AGCCTT-3'. Probe sequence 3 was obtained with a 16S primer of sequence5'GCT CGT TGC GGG ACT TAA CCC ACC AT-3'. Sequencing with these primerswas performed as described for previous examples.

The following three sequenoes were characterized by the criteriadescribed in Example 1 and were shown to be specific for the genusLegionella. The phylogenetically nearest neighbors Escherichia coli,Pseudomonas aeruginosa, Vibrio parahaemolyticus and Acinetobactercalcoaceticus were used as comparisons with sequence from Legionellaspecies.

1. TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC

2. GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC

3. CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG.

Sequence 1, from 16S rRNA, is 40 bases in length and has a Tm of 72° C.Sequence 2, from 23S rRNA, is 42 bases in length and has a Tm of 73° C.Sequence 3, from 16S rRNA, is 40 bases in length and has a Tm of 68° C.These sequences are capable of hybridizing to RNA of the genusLegionella in the regions corresponding respectively to, 630-75 of E.coli 16S rRNA; 350-395 of E. coli 23s rRNA; and 975-1020 of E. coli 16SrRNA. When mixed together the probes had a combined average Tm of 73° C.Analysis on polyacrylamide gels showed that each probe was the correctlength and sequence analysis demonstrated that each was the correctsequence of bases.

When the three probes were mixed and used in a hybridization assay, theywere found to be specific for the genus Legionella (Tables 29 and 30)and did not cross react with other respiratory pathogens or with anyselected organism from the phylogenetic tree (Tables 31 and 32). Use ofmore than one probe, i.e., a mixture of probes, can result in increasedassay sensitivity and/or in an increase in the number of non-viralorganisms to be detected.

                  TABLE 29                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA                                                   PROBES TO HOMOLOGOUS TARGET rRNA                                                              plus rRNA                                                                              minus rRNA                                           ______________________________________                                        Legionella probe                                                                              80%      1.0%                                                 ______________________________________                                    

                  TABLE 30                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA                                                   PROBES TO LEGIONELLA SPECIES                                                  Organism      ATCC#       % Probes Bound                                      ______________________________________                                        L. anisa      35292       42.0                                                L. bozemanii  33217       58.0                                                L. cherrii    35252       69.0                                                L. dumoffii   33279       57.0                                                L. erythra    CDC#9P1W044C                                                                              26.0                                                L. feeleii    35303       59.0                                                L. hackeliae  35250       47.0                                                L. jamestowniensis                                                                          35298       20.0                                                L. jordanis   33623       50.6                                                L. longbeachae                                                                              33484       48.0                                                L. maceachernii                                                                             35300       25.0                                                L. micdadei   33704       38.0                                                L. oakridgensis                                                                             33761       44.0                                                L. parisiensis                                                                               9060       69.0                                                L. pneumophila 1*                                                                            6736       75.0                                                L. pneumophila 2          64.0                                                L. pneumophila 3          73.0                                                L. pneumophila 4          73.0                                                L. pneumophila 5          78.0                                                L. pneumophila 6          75.0                                                L. pneumophila 7          73.0                                                L. pneumophila 8          63.0                                                L. pneumophila 11         75.0                                                L. rubrilucens                                                                              35304       12.0                                                L. sainthelensi                                                                             35248       61.0                                                L. sainticrucis                                                                             35301       24.0                                                L. spiritensis                                                                              CDC#MSH9    55.0                                                L. steigerwaltii                                                                             7430       56.0                                                L. wadsworthii                                                                              33877       37.0                                                ______________________________________                                         *The numbers 1-8 and 11 are serotypes of L. pneumophila.                 

                  TABLE 31                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                         RESPIRATORY PATHOGENS                                                         Organisms         ATCC#   % Probe Bound                                       ______________________________________                                        Corynebacterium xerosis                                                                         373     2.1                                                 Haemophilus influenzae                                                                          19418   2.3                                                 Klebsiella pneumoniae                                                                           23357   2.0                                                 Mycoplasma pneumoniae                                                                           15531   2.3                                                 Neisseria meningitidis                                                                          13090   2.2                                                 Pseudomonas aeruginosa                                                                          25330   1.2                                                 Propionibacterium acnes                                                                         6919    1.6                                                 Streptococcus pneumoniae                                                                        6306    0.8                                                 Staphylococcus aureus                                                                           25923   1.6                                                 ______________________________________                                    

                  TABLE 32                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                         A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                             Organisms         ATCC#   % Probe Bound                                       ______________________________________                                        Acinetobacter calcoaceticus                                                                     33604   1.4                                                 Branhamella catarrahalis                                                                        25238   2.0                                                 Bacillus subtilis 6051    1.9                                                 Bacteroides fragilis                                                                            23745   2.2                                                 Campylobacter jejuni                                                                            33560   1.2                                                 Chromobacterium violaceum                                                                       29094   1.3                                                 Clostridium perfringens                                                                         13124   1.9                                                 Deinoccoccus radiodurans                                                                        35073   1.8                                                 Derxia gummosa    15994   2.0                                                 Enterobacter aerogenes                                                                          13048   1.4                                                 Escherichia coli  11775   1.2                                                 Mycoplasma hominis                                                                              14027   1.1                                                 Proteus mirabilis 29906   1.4                                                 Pseudomonas cepacia                                                                             11762   1.1                                                 Rahnella aquatilis                                                                              33071   1.7                                                 Rhodospirillum rubrum                                                                           11170   2.0                                                 Streptococcus mitis                                                                             9811    2.0                                                 Vibrio parahaemolyticus                                                                         17802   2.0                                                 Yersinia enterocolitica                                                                         9610    1.2                                                 ______________________________________                                    

Three additional probe sequences (numbered 4-6) specific for the genusLegionella were obtained by utilizing two primers complementary toconserved regions on 23S rRNA. Sequence 4 was made from a 23S primerwith the sequence 5'-CCT TCT CCC GAA GTT ACG G-3'. Probe sequences 5 and6 were made from a 23S primer of sequence 5'-AAG CCG GTT ATC CCC GGG GTAACT TTT-3". Sequencing with these primers was performed as described forprevious examples.

The following three sequences were characterized by the criteriapreviously described and were shown to be specific for the genusLegionella. The phylogenetically nearest neighbors Escherichia coli,Pseudomonas aeruginosa, Vibrio parahaemolyticus and Actinetobactercalcoaceticus were used for comparisons with sequences from Legionellaspecies.

4. GCG GTA CGG TTC TCT ATA AGT TAT GGC TAG C

5. GTA CCG AGG GTA CCT TTG TGC T

6. CAC TCT TGG TAC GAT GTC CGA C

Probe 4, complementary to 23S rRNA in the region corresponding to bases1585-1620 of E. coli 23S rRNA, is 31 bases long and has a Tm of 67° C.Probe 5, complementary to 23S rRNA in the region corresponding to bases2280-2330 of E. coli 23S rRNA, is 22 bases long and has a Tm of 66° C.Probe 6, complementary to 23S rRNA in the same region as Probe 5, is 22bases long and has a Tm of 63° C.

When the three probes were mixed with probe 3 above and used in ahybridization assay as described for probes 1-3, they were found to bespecific for the genus Legionella (Table 33) and did not cross reactwith other respiratory pathogens or with any selected organism from thephylogenetic tree (Tables 34 and 35). Using more than one probe, i.e., amixture of probes, can improve assay sensitivity and/or increase thenumber of non-viral organisms detected.

                  TABLE 33                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                         LEGIONELLA SPECIES                                                            Organism      ATCC#        % Probes Bound                                     ______________________________________                                        L. anisa      35292        29.6                                               L. bozemanii  33217        35.5                                               L. cherrii    35252        29.2                                               L. dumoffii   33279        26.0                                               L. erythra    35303        32.0                                               L. feelii     CDC#9P1WO44C 32.0                                               L. hackeliae  35250        39.0                                               L. jamestowniensis                                                                          35298        31.2                                               L. jordanis   33623        25.7                                               L. longbeachae                                                                              33434        27.6                                               L. maceahernil                                                                              35300        39.3                                               L. micdadei   33204        31.0                                               L. oakridgensis                                                                             33761        24.4                                               L. parisiensi 35299        31.2                                               L. pneumophila 1*                                                                           33153        40.0                                               L. pneumophila 2                                                                            33154        38.5                                               L. pneumophila 3                                                                            33155        44.6                                               L. pneumophila 4                                                                            33156        48.6                                               L. pneumophila 5                                                                            33216        32.0                                               L. pneumophila 6                                                                            33215        43.0                                               L. pneumophila 7                                                                            33823        29.5                                               L. pneumophila 8                                                                            35096        37.6                                               L. pneumophila 11                                                                           43130        44.5                                               L. rubrilucens                                                                              35304        30.1                                               L. sainthelensis                                                                            35248        27.0                                               L. sainticrucis                                                                             35301        22.0                                               L. spiritensis                                                                              CDC#MSH9     40.5                                               L. steigerwaltii                                                                            35302        31.7                                               L. wadsworthii                                                                              33877        30.0                                               ______________________________________                                         *The numbers 1-8 and 11 are serotypes of L. pneumophila.                 

                  TABLE 34                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                         RESPIRATORY PATHOGENS                                                         Organisms         ATCC#   % Probe Bound                                       ______________________________________                                        Corynebacterium xerosis                                                                          373    0.13                                                Haemophilum influenzae                                                                          19418   0.12                                                Klebsiella pneumoniae                                                                           23357   0.13                                                Neisseria meningitidis                                                                          13090   0.14                                                Pseudomonas aeruginosa                                                                          25330   0.13                                                Propionibacterium acnes                                                                          6919   0.11                                                Streptococcus pneumoniae                                                                         6306   0.08                                                Staphylococcus aureus                                                                           25923   0.15                                                ______________________________________                                    

                  TABLE 35                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                         A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                             Organisms         ATCC#   % Probe Bound                                       ______________________________________                                        Acinetobacter calcoaceticus                                                                     33604   0.12                                                Branhamella catarrahalis                                                                        25238   0.13                                                Bacillus subtilis  6051   0.09                                                Bacteroides fragilis                                                                            23745   0.12                                                Campylobacter jejuni                                                                            33560   0.06                                                Chromobacterium violaceum                                                                       29094   0.33                                                Clostridium perfringens                                                                         13124   0.07                                                Deinoccoccus radiodurans                                                                        35073   0.11                                                Derxia gummosa    15994   0.15                                                Enterobacter aerogenes                                                                          13048   0.26                                                Escherichia coli  11775   0.09                                                Mycoplasma hominis                                                                              14027   0.09                                                Proteus mirabilis 29906   0.09                                                Pseudomonas cepacia                                                                             17762   0.20                                                Rahnella aquatilis                                                                              33071   0.15                                                Rhodospirillum rubrum                                                                           11170   0.13                                                Streptococcus mitis                                                                              9811   0.07                                                Vibrio parahaemolyticus                                                                         17802   0.11                                                Yersinia enterocolitica                                                                          9610   0.19                                                ______________________________________                                    

EXAMPLE 11

Chlamydia are gram-negative, non-motile, obligate intracellularbacteria. The species C. trachomatis is associated with endemic trachoma(the most common preventable form of blindness), inclusionconjunctivitis and lymphogranuloma venereum (LIV). It is a major causeof nongonococcal urethritis in men and may cause cervicitis and acutesalpingitis in women. Eye disease or chlamydial pneumonia may develop innewborns passing through the infected birth canal.

There are several methods known in the art for identification of C.trachomatis in the ufogenital tract, for example, by directimmunofluorescent staining or enzyme immunoassay of clinical specimens.The method of choice, however, remains culture of the organism inoycloheximide treated McCoy calls. Cell culture is followed bymorphological or fluorescent antibody staining for confirmation of theorganism's identity.

The inventive oligonucleotide sequences described below, when used asprobes in nucleic acid hybridization assay, accurately identifyChlamydia trachomatis isolates. This assay test is equal in sensitivityto culture or antibody tests and, in the case of culture, significantlyshortens the time to identification, and thus, diagnosis.

The use of probes to identify and distinguish between members of thespecies is novel and inventive. Indeed, Kingsbury, D. T., and E. Weiss,1968 J. Bacteriol. 96: 1421-23 (1968); Moulder, J. W., ASM News, Vol.50,No.8, (1984) report a 10% DNA homology between C. trachomatis and C.psittaci. Moreover, these reports show that different C. trachomatisstrains differ in DNA hemology. Weisberg, W. G. et. al, J. Bacteriol.167:570-574 (1986) published the 16S rRNA sequences of C. psittaci andnoted that C. trachomatis and C. psittaci share a greater than 95% rRNAhemology. From these reports, it may be inferred that it would bedifficult to invent (1) probes capable of hybridizing to all strains ofC. trachomatis; and (2) probes capable of distinguishing between C.trachomatis and C. psittaci. The following probes accomplish bothobjectives.

Ten probe sequences specific for Chlamydia trachomatis were made usingseven unique primers complementary to conserved regions of both 16S and23S rRNA. Probe sequence 1 was obtained from a 16S primer of sequence5'-TCT ACG CAT TTC ACC GCT ACA C-3'. Probe sequence 2 was obtained witha 16S primer of sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'.Sequences 3 and 4 were obtained using a 16S primer with the sequence5'-GGC CGT TAC CCC ACC TAC TAG CTA AT-3'. Probe sequences 5 and 6 wereobtained with a 23S primer of sequence 5'-CTT TCC CTC ACG GTA-3'. Probesequences 7 and 8 were obtained with a 23S primer of sequence 5'-CCT TCTCCC GAA GTT ACG G-3'. Probe sequence 9 was obtained with a 23S primer ofsequence 5'-TCG GAA CTT ACC CGA CAA GGA ATT TC-3'. Probe sequence 10 wasobtained with a primer of sequence 5'-CTA CTT TCC TGC GTC A-3'.

The following ten sequences were characterized using the criteriadescribed in Example 1 and were shown to be specific for the rRNA ofChlamydia trachomatis. The phylogenetically nearest neighbor Chlamydiapsittaci was used for comparison with Chlamydia trachomatis sequence.

1. CCG ACT CGG GGT TGA GCC CAT CTT TGA CAA

2. TTA CGT CCG ACA CGG ATG GGG TTG AGA CCA TC

3. CCG CCA CTA AAC AAT CGT CGA AAC AAT TGC TCC GTT CGA

4. CGT TAC TCG GAT GCC CAA ATA TCG CCA CAT TCG

5. CAT CCA TCT TTC CAG ATG TGT TCA ACT AGG AGT CCT GAT CC

6. GAG GTC GGT CTT TCT CTC CTT TCG TCT ACG

7. CCG TTC TCA TCG CTC TAC GGA CTC TTC CAA TCG

8. CGA AGA TTC CCC TTG ATC GCG ACC TGA TCT

9. CCG GGG CTC CTA TCG TTC CAT AGT CAC CCT AAA AG

10. TAC CGC GTG TCT TAT CGA CAC ACC CGC G

Seguence 1, from 16S rRNA, is 30 bases in length and has a Tm of 66° C.Sequence 2, from 16S rRNA, is 32 bases in length and has a Tm of 67° C.Sequence 3, from 16S rRNA, is 39 bases in length and has a Tm of 70° C.Sequence 4, from 16S rRNA, is 33 bases in length and has a Tm of 69° C.Sequence 5, from 23S rRNA, is 41 bases in length end has a Tm of 71° C.Sequence 6, from 23S rRNA, is 30 bases in length and has a Tm of 72° C.Sequence 7, from 23S rRNA, is 33 bases in length and has a Tm of 72° C.Sequence 8, from 23S rRNA, is 30 bases in length and has a Tm of 71° C.Sequence 9, from 23S rRNA is 35 bases in length and has a Tm of 74° C.Sequence 10 is 28 bases in length and has a Tm of 72° C.

The reactivity and specificity of the probes was tested hybridizationassays. ³² P-end-labeled oligonucleotide probes 1 and 2 were mixed withpurified RNA or RNA released from at least 10⁷ organisms in 0.55 ml of41% diisobutyl sulfosuccinate, 3% sodium dodecyl sulfate, 0.03M sodiumphosphate pH 6.8, 1 mM EDTA, 1 mM EGTA at 60° C. (probe 1) or 64° C.(probe 2) for 1 hour. Hybrids were bound to hydroxyapatite as describedin previous examples and the amount of radioactivity bound wasdetermined by scintillation counting. Table 36 shows that probes 1 and 2hybridize well to all serotypes of C. trachomatis tested. Probe 1 doesnot react with any strain of C. trachomatis tested and probe 2 does notreact with two of the strains. Probe 2 does react with the ovinepolyarthritis strain of C. psittaci, an organism which is not known toinfect humans. Table 37 demonstrates the reactivity and specificity ofprobes 3-9 when ¹²⁵ I-labeled and used as a mix. In this case, thehybrids were bound to cationic magnetic particles as described in Arnoldet al., U.S. patent application Ser. No. 020,866 filed Mar. 2, 1987.These probes hybridize well to all strains of C. trachomatis tested andnot to any strains of C. psittaci. Probes 3-9 were further testedagainst a panel of organisms commonly found in the urogenital tract(Table 38) and a phylogenetic cross section of organisms (Table 39). Inall cases, the probes were shown to be specific. Probe 10 is 25%non-homologous to C. psittaci and also should be specific for C.trachomatis.

                  TABLE 36                                                        ______________________________________                                        HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 1 AND 2                         TO CHLAMYDIA RNA                                                                                   % Probe Bound                                            Organism          ATCC#    Probe 1  Probe 2                                   ______________________________________                                        Chlamydia trachomatis serotype C                                                                VR578    22       39                                        Chlamydia trachomatis serotype E                                                                VR348B   27       48                                        Chlamydia trachomatis serotype G                                                                VR878    20       44                                        Chlamydia trachomatis serotype I                                                                VR880    20       42                                        Chlamydia trachomatis serotype K                                                                VR887    28       45                                        Chlamydia psittaci guinea pig                                                                   VR813    1.2      1.4                                       conjunctivitis strain                                                         Chlamydia psittaci ovine                                                                        VR656    1.0      3.0                                       abortion strain                                                               Chlamydia psittaci ovine poly-                                                                  VR619    1.1      35.3                                      arthritis strain                                                              ______________________________________                                    

                  TABLE 37                                                        ______________________________________                                        HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9                             WITH CHLAMYDIA rRNA                                                           Organism    Serovar  ATCC#   Ratio Counts Bound*                              ______________________________________                                        C. trachomatis                                                                            A                689                                              C. trachomatis                                                                            B                560                                              C. trachomatis                                                                            Ba               1066                                             C. trachomatis                                                                            C        VR548   962                                              C. trachomatis                                                                            D                1192                                             C. trachomatis                                                                            E        VR348   1022                                             C. trachomatis                                                                            F                391                                              C. trachomatis                                                                            G        VR878   874                                              C. trachomatis                                                                            H                954                                              C. trachomatis                                                                            I        VR880   943                                              C. trachomatis                                                                            J                482                                              C. trachomatis                                                                            K        VR887   999                                              C. trachomatis                                                                            L1               638                                              C. trachomatis                                                                            L2               501                                              C. trachomatis                                                                            L3       VR903   821                                              C. psittaci          VR125   1.6                                              C. psittaci          VR629   0.9                                              C. psittaci          VR656   1.3                                              C. psittaci          VR813   1.2                                              ______________________________________                                         ##STR5##                                                                 

                  TABLE 38                                                        ______________________________________                                        HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9                             TO ORGANISMS FOUND IN THE UROGENITAL TRACT.                                   Organism         ATCC#    Ratio Counts Bound*                                 ______________________________________                                        Achromobacter xylosoxidans                                                                     27061    1.9                                                 Acinetobacter lwoffii                                                                          15309    1.2                                                 Branhamella catarrhalis                                                                        25238    1.2                                                 Candida albicans 18804    2.4                                                 Flavobacterium meningosepticum                                                                 13253    1.1                                                 Gardnerella vaginalis                                                                          14018    1.3                                                 Lactobacillus acidophilus                                                                       4356    0.8                                                 Listeria monocytogenes                                                                         15313    0.7                                                 Mycobacterium smegmatis                                                                        14468    1.1                                                 Moraxella osloensis                                                                            19976    1.3                                                 Neisseria gonorrhoeae                                                                          19424    2.3                                                 Pasteurella multocida                                                                           6529    1.0                                                 Peptostreptococcus anaerobius                                                                  27337    1.2                                                 Streptococcus agalactiae                                                                       13813    4.0                                                 Streptococcus faecalis                                                                         19433    2.6                                                 ______________________________________                                         ##STR6##                                                                 

                  TABLE 39                                                        ______________________________________                                        HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9                             TO PHYLOGENETICALLY DIVERSE ORGANISMS.                                        Organism         ATCC#   Ratio Counts Bound*                                  ______________________________________                                        Bacillus subtilis                                                                               6051   2.2                                                  Bacteroides fragilis                                                                           23745   1.6                                                  Campylobacter jejuni                                                                           33560   1.4                                                  Chromabacterium violaceum                                                                      29094   1.4                                                  Deinococcus radiodurans                                                                        35073   1.8                                                  Derxia gummosa   15994   1.3                                                  Enterobacter aerogenes                                                                         13048   1.9                                                  Escherichia coli 11775   1.9                                                  Mycoplasma hominis                                                                             14027   1.3                                                  Pseudomonas cepacia                                                                            17762   2.2                                                  Proteus mirabilis                                                                              29906   2.2                                                  Rahnella aquatilis                                                                             33071   1.9                                                  Rhodospirillum rubrum                                                                          11170   1.9                                                  Vibrio parahaemolyticus                                                                        17802   2.0                                                  Yersinia enterocolitica                                                                         9610   2.5                                                  ______________________________________                                         ##STR7##                                                                 

EXAMPLE 12

Campylobacters are motile, microaerophllic, gram negative curved rods.The genus is quite diverse and distinct from other genera. Although thegenus is well defined, some revision is occurring at the species level(Romaniuk, P. J. et al., J. Bacteriol. 169:2137-2141 (1987). ThreeCampylobacter species, Campylobacter jejuni, C. coli and C. laridis,cause enteritis in humans. The disease includes diarrhea, fever, nausea,abdominal pain and in some cases, vomiting. These organisms cause anestimated 2 million infections per year in the United States (estimatebased on the number of Salmonella and Shigella induced cases ofdiarrheal disease). Other members of the genus cause septicemias inhumans and abortion and infertility in sheep and cattle.

Diagnosis of Campylobacter enteritis is currently dependent upon growthand isolation of the organism in culture, followed by a number ofbiochemical tests. Optimum growth of campylobacters requires specialconditions such as low oxygen tension and high temperature (42° C.). Nosingle set of conditions is recommended for isolation of allCampylobacter species.

The oligonucleotide sequences listed below, when used in a hybridizationassay, hybridize to the 16S rRNA of the Campylobacter species ofinterest. The present invention has significant advantages over theprior art methods of detection of Campylobacter because one probe candetect all Campylobacters of interest; the other two probes detect theenteric Campylobacters and one can detect human isolates ofCampylobacter. In addition, the probes have advantages over the priorart in terms of ease of the assay and greatly reduced time toidentification and therefore, diagnosis.

The four probes which hybridize to the 16S rRNA of Campylobacter speciesof interest were constructed using three unique primers complementary to16S rRNA. Sequences 1 and 2 were made using a 16S primer with thesequence 5'-GTA TTA CCG CGG CTG CTG GCA C-3'. Sequence 3 was made usinga 16S primer with the sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'.Sequence 4 was made with a 16S primer with the sequence 5'-GCT CGT TGCGGG ACT TAA CCC AAC AT-3'.

The following sequences were characterized and shown to hybridize toCampylobacter jejuni, C. coli and C. laridis. The phylogeneticallynearest neighbors Vibrio parahaemolyticus and Wollinella succinogeneswere used for comparison with the campylobacter sequences.

1. CGC TCC GAA AAG TGT CAT CCT CC

2. CCT TAG GTA CCG TCA GAA TTC TTC CC

3. GCC TTC GCA ATG GGT ATT CTT GGT G

4. GGT TCT TAG GAT ATC AAG CCC AGG

Sequence 1, from 16S rRNA, is 23 bases in length and has a Tm of 65° C.Sequence 2, from 168 rRNA, is 26 bases in length and has a Tm of 64° C.Sequence 3, from 16S rRNA, is 25 bases in length and has a Tm of 66° C.Sequence 4, from 16S rRNA, is 24 bases in length and has a Tm of 61° C.Sequence 1 is capable of hybridizing in the region corresponding tobases 405-428 of E. coli 16S rRNA; Sequence 2 is capable of hybridizingin the region corresponding to bases 440-475 of E. coli 16S rRNA;Sequence 3 is capable of hybridizing in the region corresponding tobases 705-735 of E. coli 16S rRNA; Sequence 4 is capable of hybridizingin the region corresponding to bases 980-1010 of E. coli 16S rRNA.

The reactivity and specificity of the probes for campylobacter wastested in hybridization assays. ³² P-end-labeled oligonucleotide probeswere mixed with purified RNA or RNA released from cells in 0.1% sodiumdodecyl sulfate. 0.5 ml of hybridization solution (41% diisobutylsulfosuccinate, 30 mM sodium phosphate, pH 6.8, 0.7% sodium dodecylsulfate, 1 mM EDTA, 1 mM EGTA) was added and the mixture incubated at60° C. for 1 to 1.5 hour. Following incubation, 2 to 2.5 ml ofseparation solution (2% hydroxyapatite, 0.12M sodium phosphate, pH 6.8,0.02% sodium dodecyl sulfate) was added and the mixture incubated at 60°C. for five minutes. The sample was centrifuged and the supernatantremoved. 2.5 ml of wash solution (0.12M sodium phosphate, pH 6.8, 0.02%sodium dodecyl sulfate) was added and the sample mixed, centrifuged andthe supernatant removed. The radioactivity bound to the hydroxyapatitewas determined by scintillation counting.

Table 40 indicates that the probes hybridize well to the Campylobacterspecies of interest, C. jejuni, C. coli, and C. laridis. Probe 1 detectsall of the Campylobacter species tested, probes 2 and 4 detect only theenteric campylobacters, and probe 3 detects all of the Campylobacterspecies except C. sputorum, an organism isolated from cattle. Thus allof the probes are useful for identifying campylobecter in stool samples.The choice of which probe to use for other applications would dependupon the level of specificity required (i.e., enteric campylobacters, orall Campylobacter species).

                  TABLE 40                                                        ______________________________________                                        HYBRIDIZATION OF CAMPYLOBACTER PROBES 1-4                                     TO CAMPYLOBACTER SPECIES.                                                                      % Probe Bound (*)                                            Organism      ATCC#   1       2    3      4                                   ______________________________________                                        Campylobacter coli                                                                          33559   64      70   52     49                                  C. fetus      27374   68      0.1  66     0.5                                 subsp. fetus                                                                  C. fetus      19438   66      0.7  54     1.2                                 subsp. venerealis                                                             C. jejuni     33560   63      76   51     56                                  C. laridis    35221   74      73   64     52                                  C. sputorum   33562   71      3.0  2.5    0                                   subsp. bubulus                                                                ______________________________________                                         (*) % Probe Bound = cpm bound to hybroxyapatitecpm bound when no RNA          present/total cpm used in the assay                                      

Table 41 shows that the probes do not hybridize to closely relatedorganisms or organisms found in the gastrointestinal tract.

                  TABLE 41                                                        ______________________________________                                        HYBRIDIZATION OF CAMPYLOBACTER PROBES 1-4 TO                                  CLOSELY RELATED ORGANISMS AND ORGANISMS FOUND IN                              THE GASTRO-INTESTINAL TRACT.                                                                      % Probe Bound (*)                                         Organism         ATCC#   1       2   3     4                                  ______________________________________                                        Bacteroides fragiles                                                                           25285   0       0.2 0.7   0                                  Escherichia coli 11775   1.3     0.5 0.5   0                                  Salmonella typhimurium                                                                         14028   0       0   0.3   0                                  Shigella boydii  29929   0       0.2 0.5   0                                  Shigella dysenteriae                                                                           13313   0       0.7 0.2   0                                  Shigella flexneri                                                                              29903   0       0   0.5   0                                  Shigella sonnei  29930   0       0   0.1   0                                  Vibrio parahaemolyticus                                                                        17802   0       1.9 0.1   0                                  Wollinella succinogenes                                                                        29543   0.4     2.1 2.2   0                                  Yersinia pseudotuberculosis                                                                    29833   0.6     0.2 1.7   0.3                                ______________________________________                                         (*) % probe bound = cpm bound to hydroxyapatitecpm bound when no RNA          present/total cpm used in the assay                                      

The probes specific for the enteric Campylobacters, probes 2 and 4, werefurther tested and shown not to react with rRNAs of other organismsfound in the gastrointestinal tract.

                  TABLE 42                                                        ______________________________________                                        HYBRIDIZATION OF CAMPYLOBACTER PROBES 2 AND 4 TO                              ORGANISMS FOUND IN THE GASTROINTESTINAL TRACT.                                                    % Probe Bound (*)                                         Organism         ATCC#    Probe 2 Probe 4                                     ______________________________________                                        Citrobacter diversus                                                                           27156    0       0                                           Clostridium perfringens                                                                        13124    0       0                                           Enterobacter cloacae                                                                           13047    0       0                                           Klebsiella pneumoniae                                                                          23337    0       0.5                                         Proteus mirabilis                                                                              25933    0       0                                           Serratia marcescens                                                                            13880    0       0                                           Staphylococcus aureus                                                                          e12600                                                       Staphylococcus epidermidis                                                                     14990    0       0.3                                         Streptococcus bovis                                                                            33317    0       0                                           ______________________________________                                         (*) % probe bound = cpm bound to hydroxyapatitecpm bound when no RNA          present/total cpm used in the assay                                      

EXAMPLE

Streptococci are gram positive, oxidase negative coccoid bacteria. Thegenus has been divided into 18 groups, A-R, on the basis ofgroup-specific carbohydrates. Group D streptococci are furthersubdivided into the enteroccocci (S. faecium, S. faecalis, S. avium andS. gallinarum and the non-enterococci S. bovis and S. equinus. S.faecium, S. faecalis and S. avium are considered the medically importantenteroccocoi. Some species of streptocoocus are human pathogens; othersare normal flora in the mouth and intestine but are capable of causingdisease when introduced to other sites. Two examples are S. faecium andS. faecalis which are normally found in the intestine but may spread tocause bacteremia, wound infections, and as many as 10% of the urinarytract infections in the United States.

Current methods of detection of enterococci require culture of thespecimen for 18-72 hours followed by a battery of biochemical tests. Theoligonucleotide sequence shown below, when used in a hybridizationassay, accurately detects Streptococcus faecalis, S. avium, and S.faecium. The inventive probe does not cross react with otherStreptococci or Staphylococci which are very closely related in DNAhomology. (Kiepper-Baez, 1981, 1982, Schliefer 1984.) The currentinvention also reduces the number of tests which must be run on a sampleand greatly reduces the time to identification and thus, diagnosis. Thisrepresents a significant improvement over prior art methods.

The probe sequence was identified using a primer complementary to 16SrRNA with the sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'. Thefollowing sequence was characterized and shown to be specific for threeenterococci, S. faecium, S. faecalis and S. avium. The phylogeneticallynearest neighbors S. agalactiae, S. bovis, S. pneumonniae and S.pyogenes were used for comparison with the sequences of interest.

1. TGC AGC ACT GAA GGG CGG AAA CCC TCC AAC ACT TA

The sequence is 35 bases in length and has a Tm of 72° C. It is capableof hybridizing in the region corresponding to bases 825-860 of E. coli16S rRNA. To demonstrate the reactivity and specificity of the probe, itwas used in a hybridization assay with purified RNA or RNA released fromcells. A suspension containing at least 10⁷ cells in 2% sodium dodecylsulfate was vortexed in the presence of glass beads. 0.1 ml ofsuspension was mixed with 0.1 ml of hybridization buffer (0.96M sodiumphosphate, pH 6.8, 0.002M EDTA, 0.002M EGTA) and incubated at 65° C. for2 hours. After incubation, 5 ml of 2% hydoxyapatite, 0.12M sodiumphosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixturewas incubated at 65° C. for 10 minutes. The sample was centrifuged andthe supernatant removed. Five ml of wash solution (0.12M phosphatebuffer, pH 6.8, 0.02% sodium dodecyl sulfate) was added and the sampleswere vortexed, centrifuged, and the supernatant removed. The amount ofradioactivity bound to the hydroxyapatite was determined byscintillation counting. Table 43 shows that the probe reacts well withS. faecium, S. fecalis, and S. avium, and does not react with otherclosely related organisms.

                  TABLE 43                                                        ______________________________________                                        HYBRIDIZATION OF THE ENTEROCOCCUS PROBE                                       TO CLOSELY RELATED ORGANISMS.                                                 Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Staphylococcus aureus                                                                           12600   1.4                                                 Streptococcus agalactiae                                                                        13813   1.5                                                 Streptococcus avium                                                                             14025   22.7                                                Streptococcus bovis                                                                             33317   1.4                                                 Streptococcus faecalis                                                                          19433   45.3                                                Streptococcus faecium                                                                           19434   43.0                                                Streptococcus mitis                                                                              9811   1.5                                                 Streptococcus pneumoniae                                                                         6306   1.5                                                 Streptococcus pyogenes                                                                          19615   1.3                                                 ______________________________________                                    

EXAMPLE 14

Pseudomonads are gram-negative, nonsporeforming, nonfermentativebacilli. Pseudomonads are common inhabitants of soil and water andrarely infect healthy individuals. When the organisms encounter alreadycompromised patients, they can cause a variety of clinical syndromesincluding wound infections, postsurgical infections, eepticemia, infantdiarrhea and respiratory and urinary tract infections. Members of thegenus Pseudomonas are particularly important to identify in a clinicalsample because of the resistance of the organisms to antibiotics.Nucleic acid homology Studies have divided the genus into five homologyclasses known as RNA groups I-V. Eighty-three percent of all clinicalisolates of Pseudomonas are from RNA group I and Pseudomonas aeruginosais by far the most common species isolated.

Current methods of detection of pseudomonas require culture of a patientSample for 24-72 hours, followed by a battery of biochemical tests. Theoligonucleotide sequence below, when used in a hybridization assay,detects the clinically important group I pseudomonas. The presentinvention reduces the number of tests which must be run on a sample, andreduces the time to detection. This represents a significant improvementover prior art methods.

The sequence was obtained with a primer complementary to a conservedregion on 23S rRNA with the sequence 5'-CTT TCC CTC ACG GTA-3'. Thefollowing sequence was shown to detect group I pseudomonads:

    CAG ACA AAG TTT CTC GTG CTC CGT CCT ACT CGA TT             1.

The probe is 35 bases in length and has a Tm of 70° C. It is capable ofhybridizing to the RNA of group I Pseudomonas in the regioncorresponding to bases 365-405 of E. coli 23S rRNA. To demonstrate thereactivity and specificity of the probe, it was used in a hybridizationassay. ³² P-end-labeled oligonucleotide was mixed with RNA released fromat least 10⁷ organisms by standard methods in 0.48M sodium phosphate pH6.8, 1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA and incubated at65° C. for two hours. After incubation, the RNA:DNA hybrids were boundto hydroxyapatite as described for previous examples and theradio-activity bound was determined by scintillation counting. Table 44demonstrates that the probe reacted well with all 8 species of group Ipseudomonads that were tested. The probe did not react with RNA fromgroup II or group V organisms. A low reaction was seen with Pseudomonasacidovorans, a group III organism which represents <1% of all isolatesof nonfermentative bacilli from clinical samples. Table 45 demonstratesthat the probe does not react with other closely related organisms whichwere tested.

                  TABLE 44                                                        ______________________________________                                        HYBRIDIZATION OF PSEUDOMONAS GROUP I                                          PROBE TO PSEUDOMONAS RNAs                                                                                       % Probe*                                    Organism        Group     ATCC#   Bound                                       ______________________________________                                        Pseudomonas alcaligenes                                                                       I         14909   24                                          Pseudomonas aeruginosa                                                                        I         10145   83                                          Pseudomonas denitrificans                                                                     I         13867   83                                          Pseudomonas fluorescens                                                                       I         13525   82                                          Pseudomonas mendocina                                                                         I         25411   79                                          Pseudomonas pseudoalcaligenes                                                                 I         17440   78                                          Pseudomonas putida                                                                            I         12633   80                                          Pseudomonas stutzeri                                                                          I         17588   84                                          Pseudomonas cepacia                                                                           II        25416   0                                           Pseudomonas pickettii                                                                         II        27511   1.0                                         Pseudomonas acidovorans                                                                       III       15668   11                                          Pseudomonas maltophilia                                                                       V         13637   0.2                                         ______________________________________                                         *% Probe Bound = counts bound when RNA present  counts bound when no RNA      present/total counts used in the assay                                   

                  TABLE 45                                                        ______________________________________                                        HYBRIDIZATION OF PSEUDOMONAS GROUP I                                          PROBE TO RNAs OF CLOSELY RELATED ORGANISMS                                                               % Probe*                                           Organism           ATCC#   Bound                                              ______________________________________                                        Acinetobacter calcoaceticus                                                                      23055   1.6                                                Legionella pneumophila                                                                           33155   0.6                                                Moraxella phenylpyruvica                                                                         23333   0.3                                                Morganella morganii                                                                              25830   0                                                  Vibrio parahaemolyticus                                                                          17802   0.6                                                ______________________________________                                         *% Probe Bound = counts bound when RNA present  counts bound when no RNA      present/total counts used in the assay                                   

EXAMPLE 15

Examples 15-18 disclose probes for the Enterobacteriaceae, all of whichare highly related at the DNA level. Even fewer differences exist at therRNA level. For example, Proteus vulgaris 16S rRNA is 93% homologous toE. coli. These factors illustrate the difficulties associated withmaking rRNA probes specific for this group of organisms. Nevertheless,we have invented probes for Enterobacter cloacae, Proteus mirabilis,Salmonella and E. coli.

Members of the genus Enterobacter are motile, gram negative,non-sporeforming bacilli which belong in the family Enterobacteriaceae.The genus is a large and heterogeneous group. Eight species have beendefined but only 5 are clinically significant. Enterobacter cloacae andE. aerogenes are the most common isolates and are associated withgenitourinary, pulmonary, blood, central nervous system and soft tissueinfections in humans.

The current method for identifying Enterobacter cloacae from patientsamples involves culture of the specimen on agar plates for 18-24 hours,followed by a battery of biochemical tests. The oligonucleotide sequencedescribed below, when used as a probe in a nucleic acid hybridizationassay, accurately identifies Enterobacter cloacae. The present inventionreduces the number of tests which must be run on a sample, the time toidentification and therefore, diagnosis, and thus represents asignificant improvement over prior art methods.

The probe specific for Enterobacter cloacae was obtained with a primercomplementary to a conserved region of 23S rRNA with the sequence 5'-CAGTCA GGA GTA TTT AGC CTT-'3 .

The following sequence was characterized and shown to be specific for E.cloacae. The phylogenetically nearest neighbors Escherichia coli,Klebsiella pneumoniae, Proteus vulgaris, Salmonella enteritidis, andCitrobactor freundii were used as comparisons with the sequence of E.cloacae.

1. GTG TGT TTT CGT GTA CGG GAC TTT CAC CC

The probe is 29 bases in length and has a Tm of 68° C. It is capable ofhybridizing to RNA of E. cloacae in the region corresponding to bases305-340 of E. coli 23S rRNA. To demonstrate the reactivity andspecificity of the probe for E. cloacae, it was used in a hybridizationassay. ³² P-end-labeled oligonucleotide probe was mixed with RNAreleased from at least 10⁷ organisms in 1% sodium dodecyl sulfate, 0.48Msodium phosphate, pH 6.8 (0.2 ml final volume) and incubated at 60° C.for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, 0.12Msodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and themixture incubated at 60° C. for 10 minutes. The sample was centrifugedand the supernatant removed. Five ml of wash solution (0.12M sodiumphosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added, the samplevortexed, centrifuged and the supernatant removed. The amount ofradioactivity bound to the hydroxyapatite was determined byscintillation counting. The results are shown in Table 46 anddemonstrates that the probe reacts well with E. cloacae and does notreact with the RNA of closely related organisms.

                  TABLE 46                                                        ______________________________________                                        HYBRIDIZATION OF ENTEROBACTER CLOACAE PROBE                                   TO CLOSELY RELATED ORGANISMS                                                                            % Probe                                             Organisms Name    ATCC#   Bound                                               ______________________________________                                        Citrobacter freundii                                                                            8090    1.8                                                 Enterobacter aerogenes                                                                          13048   1.4                                                 Enterobacter cloacae                                                                            13047   27.                                                 Escherichia coli  11775   1.0                                                 Klebsiella pneumoniae                                                                           13883   1.7                                                 Proteus mirabilis 29906   0.9                                                 Proteus vulgaris  13315   0.6                                                 Providencia stuartii                                                                            29914   1.1                                                 ______________________________________                                    

Table 47 shows that the probe does not react with the RNA of organismsfound in urine.

                  TABLE 47                                                        ______________________________________                                        HYBRIDIZATION OF ENTEROBACTER CLOACAE                                         PROBE TO ORGANISMS FOUND IN URINE.                                                                       % Probe                                            Organisms Name     ATCC#   Bound                                              ______________________________________                                        Candida albicans   18804   0.8                                                Candida krusei     34135   0.8                                                Candida parapsilosis                                                                             22019   0.9                                                Candida tropicalis 750     1.1                                                Pseudomonas aeruginosa                                                                           10145   1.0                                                Serratia marcescens                                                                              13880   1.6                                                Staphylococcus aureus                                                                            12600   1.7                                                Staphylococcus epidermidis                                                                       14990   1.4                                                Streptococcus agalactiae                                                                         13813   2.5                                                Streptococcus faecium                                                                            19434   1.5                                                Torulopsis glabrata                                                                              2001    0.9                                                ______________________________________                                    

EXAMPLE 16

Members of the genus Proteus are motile, gram negative, non-sporeformingbacilli which belong in the family Enterobacteriaceae. Four species ofProteus have been described and three of them, Proteus mirabilis, P.vulgaris, and P. penneri, cause human disease.

The most common type of proteus infection involves the urinary tract,but septicemia, pneumonia and wound infections also occur. Proteusmirabilis is the species most often isolated and may account for up to10% of all acute, uncomplicated urinary tract infections. Species,rather than genus level identification of the causative organism isdesirable because of differential antibiotic susceptibility among thespecies.

The current method for identifying Proteus mirabilis from patientsamples involves culture of the specimen on agar plates for 18-24 hours,followed by a battery of biochemical tests. The oligonucleotide sequencedescribed below, when used as a probe in a nucleic acid hybridizationassay, accurately identifies Proteus mirabilis. The present inventionreduces the number of tests which must be run on a sample, the time toidentification and therefore, diagnosis and treatment. This represents asignificant improvement over prior art methods.

The probe specific for Proteus mirabilis was obtained with a primercomplementary to a conserved region of 23S rRNA with the sequence 5'-CAGTCA GGA GTA TTT AGC CTT-3'.

The following sequence was characterized and shown to be specific for P.mirabilis. The phylogenetically nearest neighbors Escherichia coli,Klebsiella pneumoniae, Proteus vulgaris and Salmonella enteritidis wereused as comparisons with the sequence of Proteus mirabilis.

1. CCG TTC TCC TGA CAC TGC TAT TGA TTA AGA CTC

This probe is capable of hybridizing to the RNA of P. mirabilis in theregion corresponding to base 270-305 of E. coli 23S rRNA. The probe is33 bases in length and has a Tm of 66° C. To demonstrate the reactivityand specificity of the probe for P. mirabilis, it was used in ahybridization assay. ³² P-end-labeled oligonucleotide probe was mixedwith RNA released from at least 10⁷ organisms in 1% sodium dodecylsulfate, 0.48M sodium phosphate, pH 6.8, 1 mM EDTA, 1 mM EGTA (0.2 mlfinal volume) and incubated at 64° C. for 2 hours. Following incubation,5 ml of 2% hydroxyapatite, 0.12M sodium phosphate pH 6.8, 0.02% sodiumdodecyl sulfate was aided and the mixture incubated at 64° C. for 10minutes. The sample was centrifuged and the supernatant removed. Five mlof wash solution (0.12M sodium phosphate, pH 6.8, 0.02% sodium dodecylsulfate) was added, the sample vortexed, centrifuged and the supernatantwas removed. The amount of radioactivity bound to the hydroxyapatite wasdetermined by scintillation counting. The results are shown in Table 48and demonstrate that the probe reacts well with P. mirabilis and doesnot react with 27 other closely related bacteria. Table 49 shows thatthe probe does not react with 24 other phylogenetically diverse bacteriaand two yeasts tested in the same manner as the organisms in Table 48.

                  TABLE 48                                                        ______________________________________                                        HYBRIDIZATION OF PROTEUS MIRABILIS PROBE                                      TO CLOSELY RELATED ORGANISMS                                                                             % Probe                                            Organism Name      ATCC#   Bound                                              ______________________________________                                        Citrobacter diversus                                                                             27156   1.1                                                Citrobacter freundii                                                                             8090    1.1                                                Citrobacter freundii                                                                             6750    1.0                                                Enterobacter aerogenes                                                                           13048   1.0                                                Enterobacter agglomerans                                                                         27155   1.0                                                Enterobacter cloacae                                                                             e13047  1.1                                                Enterobacter gergoviae                                                                           33028   1.0                                                Enterobacter sakazakii                                                                           29544   1.1                                                Escherichia coli   10798   1.2                                                Escherichia coli   11775   1.2                                                Escherichia coli   29417   1.2                                                Klebsiella oxytoca 13182   1.0                                                Klebsiella ozaenae 11296   1.1                                                Klebsiella planticola                                                                            33531   0.9                                                Klebsiella pneumoniae                                                                            13883   1.3                                                Klebsiella pneumoniae                                                                            23357   1.1                                                Klebsiella rhinoscleromatis                                                                      13884   1.2                                                Klebsiella terrigena                                                                             33257   1.1                                                Klebsiella travisanii                                                                            33558   1.0                                                Kluyvera ascorbata 33433   0.9                                                Proteus mirabilis  25933   69.0                                               Proteus penneri    33519   2.5                                                Proteus vulgaris   13315   1.7                                                Providencia alcalifaciens                                                                        9886    1.1                                                Providencia rettgeri                                                                             29944   1.3                                                Providencia stuartii                                                                             29914   1.1                                                Salmonella arizonae                                                                              29933   1.1                                                Salmonella enteritidis                                                                           13076   0.8                                                ______________________________________                                    

                  TABLE 49                                                        ______________________________________                                        HYBRIDIZATION OF PROTEUS MIRABILIS PROBE TO                                   PHYLOGENETICALLY DIVERSE ORGANISMS                                                                       % Probe                                            Organism Name      ATCC#   Bound                                              ______________________________________                                        Acinetobacter calcoaceticus                                                                      33604   0.8                                                Bacillus subtilis  6051    1.2                                                Bacteroides fragilis                                                                             23745   0.9                                                Branhamella catarrhalis                                                                          25238   0.7                                                Campylobacter jejuni                                                                             33560   1.0                                                Candida krusei     34135   0.8                                                Chromobacterium violaceum                                                                        29094   1.1                                                Clostridium perfringens                                                                          13124   0.9                                                Deinococcus radiodurans                                                                          35073   0.8                                                Derxia gummosa     15994   0.8                                                Hafnia alvei       13337   0.9                                                Morganella morganii                                                                              25830   0.9                                                Pseudomonas aeruginosa                                                                           10145   1.0                                                Pseudomonas cepacia                                                                              17762   0.9                                                Rahnella aquatilis 33071   0.9                                                Rhodospirillum rubrurm                                                                           11170   0.8                                                Serratia marcescens                                                                              13880   0.9                                                Serratia odorifera 33077   0.9                                                Staphylococcus aureus                                                                            e12600  0.8                                                Staphylococcus epidermidis                                                                       14990   0.8                                                Streptococcus mitis                                                                              9811    0.8                                                Streptococcus pneumoniae                                                                         e6306   0.9                                                Torulopsis glabrata                                                                              2001    0.9                                                Vibrio parahaemolyticus                                                                          17802   0.8                                                Xanthomonas maltophilia                                                                          13637   1.1                                                Yersinia enterocolitica                                                                          9610    0.8                                                ______________________________________                                    

EXAMPLE 17

Members of the genus Salmonella are motile, gram negative,non-sporeforming bacilli which belong in the family Enterobacteriaceae.All salmonellae are highly related and some microbiologists considerthem to be one species. Five subgroups have been identified usingnucleic acid homology studies and over 1400 different serotypes havebeen described. All serotypes have been implicated in human entericdisease ranging from self-limited gastroenteritis with mild symptoms, tosevere gastroenteritis with bacteremia, to typhoid fever, a potentiallylife-threatening illness. S. cholerasuis, S. paratyphi A and S. typhiare the serotypes most often associated with severe disease andbacteremia. Diagnosis of Salmonella-induced enteritis is dependent upondetection of the organism in stool samples. Because infection occursprimarily by ingestion of contaminated milk, food and water, methods foridentifying Salmonella in these products before release to consumers iscritical.

Current methods for detection of members of the genus Salmonella involveculture of the specimen for 1-3 days on selective media followed by abattery of biochemical tests. Often an enrichment step is needed toisolate Salmonella from clinical samples or food products. Theoligonucleotide sequehces shown below, when used in a hydridizationassay, accurately identify members of the genus Salmonella. The presentinventive probes are specific for all members of the genus and do notreact with the other closely related Enterobacteriaceae genera. Theseinventive probes reduce the number of tests which must be run on asample and greatly reduce the time to identification. This represents asignificant improvement over prior art methods.

The probes specific for the genus Salmonella were obtained with twoprimers complementary to 16S and 23S rRNA. Sequence 1 was obtained usinga 16S primer with the sequence 5' TTA CTA GCG ATT CCG ACT TCA 3'.Sequence 2 was obtained using a 23S primer with the sequence 5' CAG TCAGGA GTA TTT AGC CTT 3+. The following sequences were characterized andshown to be specific for the genus Salmonella:

1. CTC CTT TGA GTT CCC GAC CTA ATC GCT GGC

2. CTC ATC GAG CTC ACA GCA CAT GCG CTT TTG TGT A

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 73° C.Sequence 2, from 23S rRNA, is 34 bases long and has a Tm of 71° C. Theseprobes are capable of hybridizing in the regions corresponding to bases1125-1155 of E. coli 16S rRNA and 335-375 of E. coli 23S rRNA,respectively. To demonstrate the reactivity end specificity of probe 1for members of the genus Salmonella, ³² P-end-labeled oligonuoleotidewas tested as a probe in a hybridization reaction. Purified RNA, or RNAreleased from at least 10⁷ organisms by standard methods, was mixed with1 ml hybridization buffer (final concentration 43% diisobutylsulfosuccinate, 60 mM sodium phosphate pH 6.8, 1 mM EDTA, 1 mM EGTA) andincubated at 72° C. for 2-12 hours. Following incubation, 5 ml ofseparation solution (2% hydroxyapatite, 0.12M sodium phosphate, pH 6.8,0.02% sodium dodecyl sulfate) was added and the sample were mixed,incubated at 72° C. for 5 minutes, centrifuged and the supernatantsremoved. Four ml of wash solution (0.12M sodium phosphate pH 6.8, 0.02%sodium dodecyl sulfate) was added and the samples were vortexed,centrifuged, and the supernatants removed. The amount of radioactivitybound to the hydroxyapatite was determined by scintillation counting.The results shown in Table 50 indicate that a combination of the twoprobes hybridized to the 5 subgroups of Salmonella and to all 31 of theserotypes which were tested.

                  TABLE 50                                                        ______________________________________                                        HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2                                    TO MEMBERS OF THE GENUS SALMONELLA                                                                   % Probe Bound                                          Subgroup                                                                             Organism          ATCC#   Probe 1                                                                             Probe 2                                ______________________________________                                        I      Salmonella choleraesuis                                                                         10708   24    40                                     I      Salmonella enteritidis                                                                          13076   15    67                                     I      Salmonella paratyphi A                                                                          9150    1.4   70                                     I      Salmonella sp. serotype                                                                         9270    40    26                                            anatum                                                                 I      Salmonella sp. serotype                                                                         12007   54    35                                            cubana                                                                 I      Salmonella sp. serotype give                                                                    9268    12    40                                     I      Salmonella sp. serotype                                                                         8326    53    33                                            heidelberg                                                             I      Salmonella sp. serotype                                                                         11646   36    46                                            illinois                                                               I      Salmonella sp. serotype                                                                         8387    35    32                                            montevideo                                                             I      Salmonella sp. serotype                                                                         29628   52    34                                            newington                                                              I      Salmonella sp. serotype                                                                         6962    3.4   36                                            newport                                                                I      Salmonella sp. serotype                                                                         15787   34    39                                            putten                                                                 I      Salmonella sp. serotype                                                                         9712    28    30                                            saintpaul                                                              I      Sairdonella sp. serotype                                                                        8400    38    43                                            senftenberg                                                            I      Salmonella sp. serotype                                                                         12004   29    29                                            simsbury                                                               I      Salmonella sp. serotype                                                                         15791   34    30                                            sloterdijk                                                             I      Salmonella ap. serotype                                                                         8391    32    41                                            thompson                                                               I      Salmonella sp. serotype                                                                         15611   35    2.6                                           vellore                                                                I      Salmonella typhi  19430   7.0   21                                     I      Salmonella typhimurium                                                                          14028   69    69                                     II     Salmonella salamae                                                                              6959    3.0   46                                     II     Salmonella sp. serotype                                                                         15793   6.6   30                                            maarssen                                                               III    Salmonella arizonae                                                                             33952   2.9   38                                     III    Salmonella arizonae                                                                             12324   5.5   42                                     III    Salmonella arizonae                                                                             29933   2.3   62                                     III    Salmonella arizonae                                                                             29934   63    12                                     III    Salmonella arizonae                                                                             12323   4.0   39                                     III    Salmonella arizonae                                                                             12325   51    1.9                                    IV     Salmonella sp. serotype                                                                         15783   5.8   8.0                                           harmelen                                                               IV     Salmonella sp. serotype                                                                         29932   7.5   40                                            ochsenzoll                                                             V      Salmonella sp. serotype                                                                         cdc1319 60    1.8                                           bongor                                                                 ______________________________________                                    

The specificity of the probes for members of the genus Salmonella wasdemonstrated with hybridization reactions containing RNA from organismsclosely related to Salmonella. The results are shown in Table 51.

                  TABLE 51                                                        ______________________________________                                        HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2                                    TO RNA OF CLOSELY RELATED ORGANISMS                                                               % Probe Bound                                             Organism        ATCC#     Probe 1 Probe 2                                     ______________________________________                                        Citrobacter freundii                                                                          6750      2.2     0                                           Edwardsiella tarda                                                                            15947     0       0                                           Enterobacter agglomerans                                                                      27155     0.6     0                                           Enterobacter cloacae                                                                          13047     0       0                                           Enterobacter sakazakii                                                                        29544     0       0                                           Escherichia coli                                                                              10798     0       0                                           Escherichia coli                                                                              29417     0       0                                           Klebsiella pneumoniae                                                                         23357     0.7     0                                           Kluyvera ascorbata                                                                            33433     0       0.5                                         Proteus mirabilis                                                                             25933     0.2     0                                           Shigella flexneri                                                                             29903     0       0                                           ______________________________________                                         *% Probe Bound = counts bound to hydroxyapatite  counts bound when no RNA     present/total counts used in assay                                       

Table 52 shows that Salmonella probes 1 and 2 do not hybridize tophylogenetically diverse organisms.

                  TABLE 52                                                        ______________________________________                                        HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2 TO                                 RNA OF A PHYLOGENETIC CROSS SECTION OF ORGANISMS                                                   % Probe Bound*                                           Organism         ATCC#     Probe 1 Probe 2                                    ______________________________________                                        Acinetobacter calcoaceticus                                                                    33604     1.1     0.1                                        Bacillus subtilis                                                                              6051      0       0.5                                        Bacteroides fragilis                                                                           23745     0.1     0                                          Branhamella catarrhalis                                                                        25238     0.9     0                                          Campylobacter jejuni                                                                           33560     0       0.2                                        Candida krusei   34135     0.4     0.3                                        Chromobacterium violaceum                                                                      29094     1.7     0                                          Clostridium perfringens                                                                        13124     0.3     0                                          Deinococcus radiodurans                                                                        35073     1.6     0.1                                        Derxia gummosa   15994     1.2     0                                          Hafnia alvei     13337     1.8     0                                          Morganelli morganii                                                                            25830     0       1.1                                        Pseudomonas aeruginosa                                                                         10145     0.5     0.7                                        Pseudomonas cepacia                                                                            17762     0       0                                          Pseudomonas maltophilia                                                                        13637     1.9     0                                          Rahnella aquatilis                                                                             33071     1.2     0.3                                        Rhodospirillum rubrum                                                                          11170     0.9     0                                          Serratia marcescens                                                                            13880     0       0                                          Serratia odorifera                                                                             33077     2.6     0.2                                        Staphylococcus aureus                                                                          e12600    0.2     0                                          Staphylococcus epidermidis                                                                     14990     0       0                                          Streptococcus nitis                                                                            9811      1.2     0.7                                        Streptococcus pneumoniae                                                                       e6306     0       0                                          Torulopsis glabrata                                                                            2001      0       0                                          Vibrio parahaemolyticus                                                                        17802     0       0.2                                        Yersinia enterocolitica                                                                        9610      0       0                                          ______________________________________                                         *% Probe Bound = Counts bound to hydroxyapatite  counts bound when no RNA     present/total counts used in assay                                       

EXAMPLE 18

Escherichia coli is a gram negative, nonsporeforming bacillus whichbelongs in the family Enterobacteriaceae. Five species of Escherichiahave been described: E. coli, which accounts for >99% of the clinicalisolates, E. hermanii, E. blattae, E. vulneris and E. fergusonii. E.coli is a leading cause of urinary tract infections, bactermia andneonatal meningitidis, and can cause a type of gastroenteritis known astraveller's diarrhea.

The current method for identifying E. coli from patient samples involvesculture of the specimen on agar plates for 18-72 hours, followed by abattery of biochemical tests on isolated colonies. The oligonucleotidesequence described below, when used as a probe in a nucleic acidhybridization assay, accurately detects E. coli even in the presence ofother organisms. The present invention reduces the number of tests whichmust be run on a sample and reduces the time to identification andtherefore diagnosis and treatment. This represents a significantimprovement over prior art methods.

The probe specific for E. coli was derived from the published E. colisequence (Brosius, et al. Proc. Natl. Acad. Sci. U.S.A. 75:4801-4805(1978)), using Proteus vulgaris (Carbon, et al., Nuc. Acids Res.9:2325-2333 (1981)), Klebsiella pneumoniae, Salmonella enteritidis,Enterobacter gergoviae and Citrobacter feundii for comparison. The probesequence is shown below.

1. GCA CAT TCT CAT CTC TGA AAA CTT CCG TGG

It hybridizes to RNA of E. coli in the region of 995-1030 of 16S rRNA.The probe is 30 bases in length and has a T_(m) of 66° C. To demonstratethe reactivity and specificity of the probe for E. coli, it was used ina hybridization assay. ³² P-end-labeled oligonucleotide probe was mixedwith two unlabeled oligonucleotides of sequence 5'-TGG ATG TCA AGA CCAGGT AAG GTT CTT CGC GTT GCA TCG-3' and 5'-CTG ACG ACA GCC ATG CAG CACCTG TCT CAC GGT TCC CGA AGG CA-3' and with purified RNA, or RNA releasedfrom cells with detergent and heat, in 1% sodium dodecyl sulfate (SDS),0.48M sodium phosphate pH 6.8, 1 mM EDTA, 1 mM EGTA (0.2 ml finalvolume) and incubated at 60° C. for 2 hours. Following incubation, 5 mlof 2% hydroxyapatite, 0.12M sodium phosphate pH 6.8, 0.02% sodiumdodecyl sulfate was added and the mixture incubated at 60° C. for 10minutes. The sample was centrifuged and the supernatant removed. Five mlof wash solution (0.12M sodium phosphate, pH 6.8, 0.02% sodium dodecylsulfate) was added, the sample vortexed, centrifuged and the supernatantwas removed. The amount of radioactivity bound to the hydroxyapatite wasdetermined by scintillation counting.

An example of a use for this probe would be to detect E. coli in urinesamples. Table 53 shows that the probe detects 7 out of 8 strains of E.coli tested. The probe also reacts with E. fergusonii, an organism whichwould only rarely be found in urine.

Table 54 shows that the probe does not react with any other genus testedexcept Shigella, another organism rarely isolated from urine. Theseresults show that the probe will be useful in detecting E. coli fromurine samples.

                  TABLE 53                                                        ______________________________________                                        HYBRIDIZATION OF E. coli TO ESCHERICHIA SPECIES                               Organism        ATCC#   % Probe Bound                                         ______________________________________                                        Escherichia coli                                                                              10798   70                                                    E. coli         11775   67                                                    E. coli         23722   58                                                    E. coli         25404   68                                                    E. coli         25922   55                                                    E. coli         29417   72                                                    E. coli         33780   0.8                                                   E. coli         35150   45                                                    E. fergusonii   35469   55                                                    E. hermanii     33650   0.7                                                   E. vulneris     33821   0.8                                                   ______________________________________                                    

                  TABLE 54                                                        ______________________________________                                        HYBRIDIZATION OF THE E. coli PROBE TO                                         CLOSELY RELATED ORGANISMS                                                     Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Citrobacter freundii                                                                            6750    0.8                                                 Citrobacter freundii                                                                            8090    0.9                                                 Citrobacter freundii                                                                            29221   0.6                                                 Citrobacter freundii                                                                            33128   0.6                                                 Enterobacter aerogenes                                                                          13048   1.2                                                 Enterobacter agglomerans                                                                        27155   0.9                                                 Enterobacter cloacae                                                                            13047   0.9                                                 Enterobacter gergoviae                                                                          33023   0.7                                                 Enterobacter sakazakii                                                                          29544   0.6                                                 Klebsiella oxytoca                                                                              13182   0.7                                                 Klabsiella pneumoniae                                                                           13883   0.7                                                 Proteus mirabilis 29906   0.7                                                 Proteus vulgaris  13315   0.8                                                 Shibella boydii   8700    76                                                  Shigella dysenteriae                                                                            13313   0.8                                                 Shigella flexneri 29903   71                                                  Shigella sonnei   29930   75                                                  ______________________________________                                    

EXAMPLE 19

The bacteria encompass a morphologically and physiologically diversegroup of unicellular organisms which occupy most natural environments.Although many bacteria are harmless or beneficial to their environmentor host, some are harmful and cause disease. The presence of anybacteria in some locations is undesirable or indicative of disease(e.g., culture media, pharmaceutical products, body fluids such asblood, urine or cerebrospinal fluid, and tissue biopsies). Low levels ofbacteria are considered acceptable in other products such as drinkingwater and food products. Accordingly, there is a need for a means fordetecting and quantitating bacteria in a sample.

The current method of detection and quantitation of total bacteria in asample requires culture on multiple types of media under differentconditions of temperature and atmosphere. To date, no single test existsto detect or quantitate all bacteria. The oligonucleotide sequencesshown below, when used in a hybridization assay, detect a broadphylogenetic cross section of bacteria. The present invention reducesthe number of tests which need to be performed and also reduces the timerequired for the assay. Comparison of the hybridization results from anunknown sample to a set of standards will allow some quantitation of thenumber of bacteria present. This represents a significant improvementover prior art methods.

The bacterial probes were designed following examination of publishedsequences of rRNA and sequences determined at Gen-Probe. The sequencesused for the comparison include Agrobacterium tumefaciens (Yang et al.,Proc. Natl. Acad. Sci. U.S.A., 82:4443, (1985), Anacystis nidulans(Tomioka and Sugiura. Mol. Gen. Genet. 191:46, (1983), Douglas andDoolittle Nuc. Acids Res. 12:3373, (1984), Bacillus subtills (Green etal., Gene 37:261. (1985), Bacillus stearothermophilus (Kop et al., DNA3:347, (1984), Bacteroides fragilis (Weisburg et al., J. Bacteriol,164:230, (1985), Chlamydia psittaci (Weisburg et al., J. Bacteriol.167:570. (1986)), Desulfovibrio desulfuricans (Oyaizu and Woese, System.Appl. Microbiol. 6:257, (1985); Escherichia coli, (Brosius et al., Proc.Natl. Acad. Sci. U.S.A. 77:201, (1980); Flavobacterium heparinum(Weisburg et al., J. Bacteriol. 164:230, (1985); Heliobacterium chlorum(Woese et al., Science 229:762, (1985); Mycoplasma PG50 (Frydenberg andChristiansen, DNA 4:127, (1985); Proteus vulgaris (Carbon et al., Nu.Acids Res. 9:2325, (1981); Pseudomonas testosteroni (Yang et al., Proc.Natl. Acad. Sci. U.S.A, 82:4443, (1985); Rcchalimaea guintana (Weisburget al., Science 230:556, (1985); Saccharomyces cerevisiae (Rubstov etal., Nuc. Acids Res. 8:5779, (1980); Georgiev et al., Nuc. Acids Res.9:6953, (1981); and human (Torczynski et al., DNA 4:283, (1985);Gonzalez et al., Proc. Natl. Acad. Sci. U.S.A.: 82:7666, (1985)).

The following sequences were shown to hybridize to a broad phylogeneticcross section of bacteria and not to yeast or human rRNA:

1. CCA CTG CTG CCT CCC GTA GGA GTC TGG GCC

2. CCA GAT CTC TAC GCA TTT CAC CGC TAC ACG TGG

3. GCT CGT TGC GGG ACT TAA CCC AAC AT

4. GGG GTT CTT TTC GCC TTT CCC TCA CGG

5. GGC TGC TTC TAA GCC AAC ATC CTG

6. GGA CCG TTA TAG TTA CGG CCG CC

7. GGT CGG AAC TTA CCC GAC AAG GAA TTT CGC TAC C

Probe 1 is 30 bases long and has a Tm of 70° C. Probe 2 is 33 bases longand has a Tm of 69° C. Probe 3 is 26 bases long and has a Tm of 67° C.Probe 4 is 27 bases long and has a Tm of 69° C. Probe 5 is 24 bases longand has a Tm of 66° C. Probe 6 is 23 bases long and has a Tm of 62° C.Probe 7 is 34 bases long and has a Tm of 66° C. Probes 1-3 hybridize to16S rRNA in the following regions, respectively, (corresponding to E.coli bases) 330-365; 675-715; and 1080-1110. Probes 4-7 hybridize to 23SrRNA in the following regions, respectively, (corresponding to E. colibases) 460-490; 1050-1080; and 1900-1960 (probes 6 and 7). Theoligonucleotides interact with regions on the rRNA which are highlyconserved among eubacteria. This means that they can be used asbacterial probes in a hybridization assay. A second use is as a tool toobtain rRNA sequence. For example, an oligonucleotide can be hybridizedto the rRNA of interest and extended with reverse transcriptase. Thesequence of the resulting DNA can be determined and used to deduce thecomplementary rRNA sequence as described in the Detailed Description ofthe Invention.

One application of the invention is to detect bacteria in urine(bacteriuria). To demonstrate the reactivity and specificity of theprobes for bacteria found in urine, they were used in hybridizationassays. ³² P-end-labeled or ¹²⁵ I-labeled oligonucleotide probes weremixed with RNA released from cells by standard methods (e.g., the sonicdisruption techniques described in Murphy et al., U.S. Pat. No.5,374,522, detergent with glass beads, or enzymatic lysis). Probe wasmixed with RNA in 0.48M sodium phosphate, pH 6.8, 1 mM EDTA, 1 mM EGTA,1% sodium dodecyl sulfate (0.2 ml final volume) and hybridized at 60° C.for 2 hours. Five ml of 2% hydroxyapatite, 0.12M sodium phosphate pH6.8, 0.02% sodium dodecyl sulfate was added and the mixture incubated at60° C. for 10 minutes. The mixture was centrifuged and the supernatantremoved. Five ml of wash solution (0.12M sodium phosphate, pH 6.8, 0.02%sodium dodecyl sulfate) was added and the sample was mixed, centrifugedand the supernatant removed. The amount of radioactivity bound to thehydroxyapatite was determined by scintillation counting. Tables 55-68demonstrate the specificity of these probes and show that a combinationof probes could be used to detect all bacteria which have been tested.

Table 55 shows that probe 1 hybridizes to the RNA of bacteria commonlyisolated from urine and does not detect yeast RNA. Table 56 shows thatprobe 1 detects phylogenetically diverse bacteria and does not hybridizeto human RNA.

                  TABLE 55                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 1                                            TO RNA OF ORGANISMS FOUND IN URINE                                            Organism         ATCC#   % Probe* Bound                                       ______________________________________                                        Candida albicans 18804   2.6                                                  Candida krusae   34135   2.2                                                  Candida parapsilosis                                                                           22019   2.9                                                  Candida tropicalis                                                                              750    2.5                                                  Citrobacter freundii                                                                            8090   69                                                   Enterobacter aerogenes                                                                         13048   70                                                   Enterobacter cloacae                                                                           13047   71                                                   Escherichia coli 11775   67                                                   Klebsiella oxytoca                                                                             13182   70                                                   Klebsiella pneumoniae                                                                          13883   72                                                   Morganella morganii                                                                            25830   66                                                   Proteus mirabilis                                                                              29906   71                                                   Proteus vulgaris 13315   67                                                   Providencia stuartii                                                                           29914   69                                                   Pseudomonas aeruginosa                                                                         10145   76                                                   Pseudomonas fluorescens                                                                        13525   73                                                   Serratia marcescans                                                                            13880   66                                                   Staphylococcus aureus                                                                          12600   57                                                   Staphylococcus epidermidis                                                                     14990   68                                                   Streptococcus agalactiae                                                                       13813   68                                                   Streptococcus faecalis                                                                         19433   51                                                   Streptococcus faecium                                                                          19434   53                                                   Torulopsis glabrata                                                                             2001   2.3                                                  Ureaplasma urealyticum                                                                         27618   54                                                   ______________________________________                                    

                  TABLE 56                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 1 TO RNAs                                    OF A PHYLOGENETIC CROSS SECTION OF ORGANISMS                                  Organism          ATCC#   % Probe* Bound                                      ______________________________________                                        Acinetobacter calcoaceticus                                                                     23055   65                                                  Bacillus subtilis  6051   73                                                  Bacteroides fragilis                                                                            23745   61                                                  Branhamella catarrhalis                                                                         25238   72                                                  Campylobacter jejuni                                                                            33560   64                                                  Chlamydia trachomatis                                                                           VR878   14                                                  Chromabacterium violaceum                                                                       29094   71                                                  Clostridium perfringens                                                                         13124   74                                                  Corynebacterium xerosis                                                                          373    38                                                  Deinococcus radiodurans                                                                         35073   47                                                  Derxia gummosa    15994   65                                                  Gardnerella vaginalis                                                                           14018   67                                                  Hafnia alvei      13337   60                                                  Lactobacillus acidophilus                                                                        4356   56                                                  Moraxella osloensis                                                                             19976   61                                                  Mycobacterium smegmatis                                                                         14468   47                                                  Mycoplasma hominis                                                                              14027   58                                                  Nesisseria gonorrhoeae                                                                          19424   58                                                  Rahnella aquatilis                                                                              33071   74                                                  Rhodospirillum rubrum                                                                           11170   73                                                  Vibrio parahaemolyticus                                                                         17802   75                                                  Human                     2.5                                                 ______________________________________                                    

Table 57 shows that Probe 2 hybridizes to the RNA of bacteria commonlyfound in urine except Ureaplasma urealyicum and does not hybridize toyeast rRNA.

                  TABLE 57                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 2                                            TO RNA OF ORGANISMS FOUND IN URINE                                            Organism         ATCC#   % Probe* Bound                                       ______________________________________                                        Candida albicans 18804   2.5                                                  Candida krusei   34135   1.8                                                  Candida parapsilosis                                                                           22019   1.6                                                  Candida tropicalis                                                                              750    1.4                                                  Citrobacter freundii                                                                            8090   61                                                   Enterobacter aerogenes                                                                         13048   57                                                   Enterobacter cloacae                                                                           13047   61                                                   Escherichia coli 11775   67                                                   Klebsiella oxytoca                                                                             13182   67                                                   Klebsiella pneumoniae                                                                          13883   51                                                   Morganella morganii                                                                            25830   69                                                   Proteus mirabilis                                                                              29906   69                                                   Proteus vulgaris 13315   69                                                   Providencia stuartii                                                                           29914   66                                                   Pseudomonas aeruginosa                                                                         10145   59                                                   Pseudomonas fluorescens                                                                        13525   58                                                   Serratia marcescens                                                                            13880   64                                                   Staphylococcus aureus                                                                          12600   60                                                   Staphylococcus epidermidis                                                                     14990   60                                                   Streptococcus agalactiae                                                                       13813   54                                                   Streptococcus faecalis                                                                         19433   37                                                   Streptococcus faecium                                                                          19434   58                                                   Torulopsis glabrata                                                                             2001   1.5                                                  Ureaplasma urealyticum                                                                         27618   3.2                                                  ______________________________________                                    

Table 58 shows that probe 2 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 58                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 2 To RNAs                                    OF A CROSS SECTION OF PHYLOGENETICALLY DIVERSE                                ORGANISMS.                                                                    Organism          ATCC#   % Probe* Bound                                      ______________________________________                                        Acinetobacter calcoaceticus                                                                     23055   76                                                  Bacillus subtilis  6051   75                                                  Bacteroides fragilis                                                                            23745   2.0                                                 Branhamella catarrhalis                                                                         25238   70                                                  Campylobacter jejuni                                                                            33560   2.5                                                 Chlamydia trachomatis                                                                           VRS78   16                                                  Chromobacterium violaceum                                                                       29094   61                                                  Clostridium perfringens                                                                         13124   66                                                  Corynebacterium xerosis                                                                          373    3.8                                                 Deinococcus radiodurans                                                                         35073   6.0                                                 Derxia gummosa    15994   61                                                  Gardnerella vaginalis                                                                           14018   2.0                                                 Hafnia alvei      13337   72                                                  Lactobacillus acidophilus                                                                        4356   50                                                  Moraxella osloensis                                                                             19976   64                                                  Mycobacterium smegmatis                                                                         14468   19                                                  Mycoplasma hominis                                                                              14027   34                                                  Neisseria gonorrhoeae                                                                           19424   71                                                  Rahnella aquatilis                                                                              33071   77                                                  Rhodospirillum rubrum                                                                           11170   1.5                                                 Vibrio parahaemolyticus                                                                         17802   73                                                  Yersinia enterocolitica                                                                          9610   76                                                  Human                     2.0                                                 ______________________________________                                    

Table 59 shows that probe 3 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 59                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNA OF                                  ORGANISMS FOUND IN URINE.                                                     Organism         ATCC#   % Probe* Bound                                       ______________________________________                                        Candida albicans 18804   1.4                                                  Candida krusei   34135   1.5                                                  Candida parapsilosis                                                                           22019   2.2                                                  Candida tropicalis                                                                              750    2.6                                                  Citrobacter freundii                                                                            8090   79                                                   Enterobacter aerogenes                                                                         13048   40                                                   Enterobacter cloacae                                                                           13047   44                                                   Escherichia coli 11775   67                                                   Klebsiella oxytoca                                                                             13182   38                                                   Klebsiella pneumoniae                                                                          13883   45                                                   Morganella morganii                                                                            25830   57                                                   Proteus mirabilis                                                                              29906   40                                                   Proteus vulgaris 13315   51                                                   Providencia stuartii                                                                           29914   54                                                   Pseudomonas aeruginoma                                                                         10145   61                                                   Pseudomonas fluorescens                                                                        13525   56                                                   Serratia marcescens                                                                            13880   54                                                   Staphylococcus aureus                                                                          12600   37                                                   Staphylococcus epidermidis                                                                     14990   20                                                   Streptococcus agalactiae                                                                       13813   34                                                   Streptococcus faecalis                                                                         19433   20                                                   Streptococcus faecium                                                                          19434   47                                                   Torulopsis glabrata                                                                             2001   1.9                                                  Ureaplasma urealyticum                                                                         27618   26                                                   ______________________________________                                    

Table 60 shows that probe 3 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 60                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNAs                                    OF A CROSS SECTION OF PHYLOGENETICALLY DIVERSE                                ORGANISMS.                                                                    Organism Name      ATCC#   % Probe Bound                                      ______________________________________                                        Acinetobacter calcoaceticus                                                                      23055   69                                                 Bacillus subtilis   6051   35                                                 Bacteroides fragilis                                                                             23745   1.2                                                Branhamella catarrhalis                                                                          25238   43                                                 Campylobacter jejuni                                                                             33560   55                                                 Chlamydia trachomatis                                                                            VR878   42                                                 Chromobacterium violaceum                                                                        29094   69                                                 Clostridium perfringens                                                                          13124   62                                                 Corynebacterium xerosis                                                                           373    23                                                 Deinococcus radiodurans                                                                          35073   30                                                 Derxia gummosa     15994   67                                                 Gardnerella vaginalis                                                                            14018   40                                                 Hafnia alvei       13337   56                                                 Lactobacillus acidophilus                                                                         4356   36                                                 Moraxella osloensis                                                                              19976   64                                                 Mycobacterium smegmatis                                                                          14468   77                                                 Mycoplasma hominis 14027   1.5                                                Neisseria gonorrhoeae                                                                            19424   26                                                 Rahnella aquatilis 33071   66                                                 Rhodospirillum rubrum                                                                            11170   51                                                 Vibrio parahaemolyticus                                                                          17802   68                                                 Yersinia enterocolitica                                                                           9610   68                                                 Human                      0.9                                                ______________________________________                                    

Table 61 shows that probe 4 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 61                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 4 TO RNA OF                                  ORGANISMS FOUND IN URINE.                                                     Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Candida albicans  18804   4.5                                                 Candida krusei    34135   2.5                                                 Candida parapsilosis                                                                            22019   2.7                                                 Candida tropicalis                                                                               750    2.5                                                 Citrobacter freundii                                                                             8090   55                                                  Enterobacter aerogenes                                                                          13048   52                                                  Enterobacter cloacae                                                                            13047   57                                                  Escherichia coli  11775   70                                                  Klebsiella oxytoca                                                                              13182   70                                                  Klebsiella pneumoniae                                                                           13883   43                                                  Morganella morganii                                                                             25830   74                                                  Proteus mirabilis 29906   74                                                  Proteus vulgaris  13315   73                                                  Providencia stuartii                                                                            29914   73                                                  Pseudomonas aeruginoga                                                                          10145   76                                                  Pseudomonas fluorescens                                                                         13525   79                                                  Serratia marcescens                                                                             13880   74                                                  Staphylococcus aureus                                                                           12600   73                                                  Staphylococcus epidermidis                                                                      14990   73                                                  Streptococcus agalactiae                                                                        13813   70                                                  Streptococcus faecalis                                                                          19433   37                                                  Streptococcus faecium                                                                           19434   63                                                  Torulopsis glabrata                                                                              2001   2.2                                                 Ureaplasma urealyticum                                                                          27618   43                                                  ______________________________________                                    

Table 62 shows that probe 4 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 62                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 4 TO RNAs                                    OF A CROSS SECTION OF PHYLOGENETICALLY DIVERSE                                ORGANISMS.                                                                    Organism Name     ATCC#   % Probe Bound                                       ______________________________________                                        Acinetobacter calcoaceticus                                                                     23055   69                                                  Bacillus subtilis  6051   55                                                  Bacteroides fragilis                                                                            23745   3.0                                                 Branhamella catarrhalis                                                                         25238   59                                                  Campylobacter jejuni                                                                            33560   65                                                  Chlamydia trachomatis                                                                           VR878   50                                                  Chromobacterium violaceum                                                                       29094   61                                                  Clostridium perfringens                                                                         13124   57                                                  Corynabacterium xerosis                                                                          373    9.5                                                 Deinococcus radiodurans                                                                         35073   63                                                  Derxia gummosa    15994   65                                                  Gardnerella vaginalis                                                                           14018   57                                                  Hafnia alvei      13337   67                                                  Lactobacillus acidophilus                                                                        4356   68                                                  Moraxella osloensis                                                                             19976   68                                                  Mycobacterium smegmatis                                                                         14468   28                                                  Mycoplasma hominis                                                                              14027   74                                                  Neisseria gonorrhoeae                                                                           19424   76                                                  Rahnella aquatilis                                                                              33071   68                                                  Rhodospirillum rubrum                                                                           11170   59                                                  Vibrio parahaemolyticus                                                                         17802   75                                                  Yersinia enterocolitica                                                                          9610   74                                                  Human                     2.8                                                 ______________________________________                                    

Table 63 shows that probe 5 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 63                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNA OF                                  ORGANISMS FOUND IN URINE.                                                     Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Candida albicans  18804   1.8                                                 Candida krusei    34135   1.7                                                 Candida parapsilosis                                                                            22019   2.2                                                 Candida tropicalis                                                                               750    1.8                                                 Citrobacter freundii                                                                             8090   39                                                  Enterobacter aerogenes                                                                          13048   38                                                  Enterobacter cloacae                                                                            13047   43                                                  Escherichia coli  11775   31                                                  Klebsiella oxytoca                                                                              13182   38                                                  Klebsiella pneumoniae                                                                           13883   66                                                  Morganella morganii                                                                             25830   50                                                  Proteus mirabilis 29906   44                                                  Proteus vulgaris  13315   52                                                  Providencia stuartii                                                                            29914   44                                                  Pseudomonas aeruginosa                                                                          10145   47                                                  Pseudomonas fluorescens                                                                         13525   25                                                  Serratia marcescens                                                                             13880   35                                                  Staphylococcus aureus                                                                           12600   26                                                  Staphylococcus epidermidis                                                                      14990   37                                                  Streptococcus agalactiae                                                                        13813   29                                                  Streptococcus faecalis                                                                          19433   14                                                  Streptococcus faecium                                                                           19434   33                                                  Torulopsis glabrata                                                                              2001   2.2                                                 Ureaplasma urealyticum                                                                          27618   73                                                  ______________________________________                                    

Table 64 shows that probe 5 detects phylogenetically diverse bacteriaand does not hybridize to human RNA.

                  TABLE 64                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNAs                                    OF A CROSS SECTION OF PHYLOGENETICALLY DIVERSE                                ORGANISMS                                                                     Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Acinetobacter calcoaceticus                                                                     23055   20                                                  Bacillus subtilis  6051   53                                                  Bacteroides fragilis                                                                            23745   44                                                  Branhamella catarrhalis                                                                         25238   22                                                  Campylobacter jejuni                                                                            33560   35                                                  Chromabacterium violaceum                                                                       29094   59                                                  Clostridium perfringens                                                                         13124   63                                                  Corynebacterium xerosis                                                                          373    1.7                                                 Deinococcus radiodurans                                                                         35073   5.7                                                 Derxia gummosa    15994   14                                                  Gardnerella vaginalis                                                                           14018   1.6                                                 Hafnia alvei      13337   44                                                  Lactobacillus acidophilus                                                                        4356   1.5                                                 Moraxella osloensis                                                                             19976   7.2                                                 Mycobacterium smegmatis                                                                         14468   39                                                  Mycoplasma hominis                                                                              14027   21                                                  Neisseria gonorrhoeae                                                                           19424   40                                                  Rahnella aquatilis                                                                              33071   55                                                  Rhodospirillum rubrum                                                                           11170   17                                                  Vibrio parahaemolyticus                                                                         17802   66                                                  Yersinia enterocolitica                                                                          9610   64                                                  Human                     1.6                                                 ______________________________________                                    

Table 65 shows that probe 6 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 65                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNA OF                                  ORGANISMS FOUND IN URINE                                                      Organism          ATCC#   % Probe Bound                                       ______________________________________                                        Candida albicans  18804   3.0                                                 Candida krusei    34135   2.0                                                 Candida parapsilosis                                                                            22019   2.2                                                 Citrobacter freundii                                                                             8090   54                                                  Enterobacter aerogenes                                                                          13048   50                                                  Enterobacter cloacae                                                                            13047   58                                                  Escherichia coli  11775   63                                                  Klebsiella oxytoca                                                                              13182   54                                                  Klebsiella pneumoniae                                                                           13883   55                                                  Morganella morganii                                                                             25830   60                                                  Proteus mirabilis 29906   64                                                  Proteus vulgaris  13315   67                                                  Providencia stuartii                                                                            29914   64                                                  Pseudomonas aeruginosa                                                                          10145   65                                                  Pseudomonas fluorescens                                                                         13525   31                                                  Serratia marcescens                                                                             13880   67                                                  Staphylococcus aureus                                                                           12600   53                                                  Staphylococcus epidermidis                                                                      14990   34                                                  Streptococcus agalactiae                                                                        13813   31                                                  Streptococcus faecium                                                                           19434   18                                                  Torulopsis glabrata                                                                              2001   2.5                                                 ______________________________________                                    

Table 66 shows that probe 6 detects some phylogenetically diversebacteria and does not hybridize to human rRNA.

                  TABLE 66                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 6 TO RNAs                                    OF A CROSS SECTION OF PHYLOGENETICALLY                                        DIVERSE ORGANISMS.                                                                                      % Probe                                             Organism          ATCC#   Bound                                               ______________________________________                                        Acinetobacter calcoaceticus                                                                     23055   73                                                  Bacteroides fragilis                                                                            23745   7.0                                                 Branhamella catarrhalis                                                                         25238   4.0                                                 Deinococcus radiodurans                                                                         35073   5.5                                                 Derxia gummosa    15994   3.0                                                 Gardnerella vaginalis                                                                           14018   2.0                                                 Hafnia alvei      13337   3.5                                                 Lactobacillus acidophilus                                                                        4356   17                                                  Moraxella osloensis                                                                             19976   62                                                  Mycoplasma hominis                                                                              14027   44                                                  Rahnella aquatilis                                                                              33071   56                                                  Yersinia enterocolitica                                                                          9610   50                                                  Human                     4.0                                                 ______________________________________                                    

Table 67 shows that probe 7 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 67                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 7 TO RNA                                     OF ORGANISMS FOUND IN URINE                                                                             % Probe                                             Organism          ATCC#   Bound                                               ______________________________________                                        Candida albicans  18804   2.1                                                 Candida krusei    34135   2.0                                                 Candida tropicalis                                                                               750    2.2                                                 Citrobacter freundii                                                                             8090   67                                                  Enterobacter aerogenes                                                                          13048   69                                                  Enterobacter cloacae                                                                            13047   78                                                  Escherichia coli  11775   75                                                  Klabsiella oxytoca                                                                              13882   79                                                  Klebsiella pneumoniae                                                                           13883   77                                                  Morganella morganii                                                                             25830   76                                                  Proteus mirabilis 29906   77                                                  Proteus vulgaris  13315   79                                                  Providencia stuartii                                                                            29914   64                                                  Pseudomonas aeruginosa                                                                          10145   76                                                  Pseudomonas fluorescens                                                                         13525   78                                                  Serratia marcescens                                                                             13880   66                                                  Staphylococcus aureus                                                                           12600   71                                                  Staphylococcus epidermidis                                                                      14990   75                                                  Streptococcus agalactiae                                                                        13813   70                                                  Streptococcus faecalis                                                                          19433   58                                                  Streptococcus faecium                                                                           19434   68                                                  Torulopsis glabrata                                                                              2001   2.4                                                 Ureaplasma urealyticum                                                                          27618   21                                                  ______________________________________                                    

Table 68 shows that probe 7 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 68                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 7 TO RNAs                                    OF A CROSS SECTION OF PHYLOGENETICALLY                                        DIVERSE ORGANISMS                                                                                       % Probe                                             Organism          ATCC#   Bound                                               ______________________________________                                        Acinetobacter calcoaceticus                                                                     23055   86                                                  Bacillus subtilis  6051   83                                                  Bacteroides fragilis                                                                            23745   69                                                  Branhamella catarrhalis                                                                         25238   74                                                  Campylobacter jejuni                                                                            33560   5.3                                                 Chlamydia trachomatis                                                                           VR878   41                                                  Chromobacterium violaceum                                                                       29094   69                                                  Clostridium perfringens                                                                         13124   68                                                  Corynebacterium xerosis                                                                          373    23                                                  Deinococcus radiodurans                                                                         35073   70                                                  Derxia gummosa    15994   69                                                  Gardnerella vaginalis                                                                           14018   68                                                  Hafnia alvei      13337   77                                                  Moraxella osloensis                                                                             19976   68                                                  Mycobacterium smegmatis                                                                         14468   64                                                  Mycoplasma hominis                                                                              14027   4.0                                                 Neisseria gonorrhoeae                                                                           19424   53                                                  Rahnella aquatilis                                                                              33071   72                                                  Rhodospirillum rubrum                                                                           11170   73                                                  Vibrio parahaemolyticus                                                                         17802   67                                                  Yersinia enterocolitica                                                                          9610   66                                                  Human                     2.2                                                 ______________________________________                                    

EXAMPLE 20

Fungi encompass a morphologically and physiologically diverse group ofsimple eucaryotic organisms. We estimate, using published sequences ofthree fungi, Neurospora crassa, Podospora, and Saccharomyces, that therRNA of fungi are 58-60% homologous to E. coli and 84-90% homologous toone another. Some fungi grow as single cells (yeasts), others asmultinuclear filaments (molds) and still others can grow as eithersingle cells or multicellular filaments (dimorphic fungi). Although manyfungi are harmless inhabitants of their environments, others are harmfuland cause disease. The presence of any fungi in some locations isundesirable or indicative of disease (e.g., culture media,pharmaceutical products, body fluids such as blood, urine orcerebrospinal fluid, and tissue biopsies). Low levels of fungi areconsidered acceptable in other products such as drinking water and foodproducts. This has created the need for a means of detecting andquantitating fungi in a sample.

The current methods for detecting and quantifying fungi involvemicroscopic examination of samples and culture on different media.Although most yeasts can be grown from clinical samples in a matter ofdays, some filamentous fungi take up to four weeks culture time, afterwhich special staining procedures, biochemical analysis and antigentests are performed. The oligonucleotide sequences below, when used in ahybridization assay, detect the five yeasts most commonly isolated inthe clinical setting, Candida albicans, Torulopsis glabrata, Candidatropicalis, Candida parapsilosis and Candida krusei. Five other fungirepresenting the Trichosporon, Blastomyces, Cryptococcus andSaccharomyces genera are also detected. The present invention allows onestep detection of these organisms and, in relation to culture, reducesthe time to identification or elimination of these fungi as the cause ofan infection. This represents a significant improvement over prior artmethods.

The four probes which hybridize to the organisms of interest wereidentified using 3 primers complementary to conserved regions on 18S or28S rRNA. Sequence 1 was obtained using an 188 primer with the sequence5'-AGA ATT TCA CCT CTG-3'. Sequence 2 was obtained using a 28S primerwith the sequence 5'-CCT TCT CCC GAA GTT ACG G-3'. Sequences 3 and 4were obtained with a 28S primer with the sequence 5'-TTCCGA CTT CCA TGGCCA CCG TCC-3'. The following sequences were characterized and shown tohybridize to fungel rRNA. The sequences of Saccharomyces cerevisiae,Saccharomyces carlsbergensis, Escherichia coli and human rRNA were usedfor comparison with the sequences of interest.

1. CCC GAC CGT CCC TAT TAA TCA TTA CGA TGG

2. CGA CTT GGC ATG AAA ACT ATT CCT TCC TGT GG

3. GCT CTT CAT TCA ATT GTC CAC GTT CAA TTA AGC AAC AAG G

4. GCT CTG CAT TCA AAC GTC CGC GTT CAA TAA AGA AAC AGG G

Sequence 1, from 18S rRNA, is 30 bases in length and has a Tm of 68° C.Sequence 2, from 23S rRNA, is 32 bases in length and has a Tm of 67° C.Sequence 3, from 23S rRNA, is 40 bases in length and has a Tm of 66° C.Sequence 4, from 23S rRNA, is 40 bases in length and has a Tm of 68° C.Sequence 1 hybridizes in the region corresponding to position 845-880 ofSaccharomyces cerevisiae 18S rRNA. Sequence 2 hybridizes in the regioncorresponding to position 1960-2000 of Saccharomyces cerevisiae 28S rRNAand sequences 3 and 4 hybridize in the region of 1225-1270 of the 28SrRNA.

To demonstrate the reactivity and specificity of these probes for fungalRNA, they were used in hybridization assays. ³² P- or ¹²⁵ I-labeledoligonucleotide probes were mixed with purified RNA or RNA released fromcells by standard lysis techniques in 0.2 ml of 0.48M sodium phosphatepH 6.8, 1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA and incubated at60° C. for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite,0.12M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was addedand the samples incubated 10 minutes at 60° C. The samples werecentrifuged and the supernatants removed. Five ml of 0.12M sodiumphosphate pH 6.8, 0.02% sodium dodecyl sulfate was added, the sampleswere mixed, centrifuged and the supernatants removed. The results areshown in Table 69. Probe 1 detects all ten fungi which were tested,probe 2 detects all six of the yeasts which were tested, probe 3 detectsfive of the six yeasts, and probe 4 detects C. krusei only. Thus probe 4could be used to detect and identify C. krusei in samples, probes 1, 2,or a combination of 3 and 4 could be used to detect the yeasts, andprobe 1 could be used to detect any of the ten organisms listed in Table69.

One potential use for these probes is to identify yeasts in urinesamples or other normally sterile body fluids. The probes werehybridized to a panel of bacteria most commonly isolated from urine andshown not to react (Table 70). Table 71 shows that the probes do nothybridize to phylogenetically diverse bacteria or to human RNA.

                  TABLE 69                                                        ______________________________________                                        HYBRIDIZATION OF YEAST PROBES TO YEAST RNA                                                       % Probe Bound                                              Organism       ATCC#   #1      #2   #3    #4                                  ______________________________________                                        Blastomyces dermatitidis                                                                     C.I.    25      1.4  1.5   1.5                                 Candida albicans                                                                             18804   40      63   56    2.0                                 C. krusei      34135   73      62   2.2   70                                  C. parapsilosis                                                                              22019   71      63   65    2.0                                 C. tropicalis   750    62      71   71    2.0                                 Cryptococcus laurentii                                                                       C.I.    43      1.4  1.5   1.5                                 Cryptococcus neoformans                                                                      C.I.    60      1.3  1.5   1.6                                 Torulopsis glabrata                                                                           2001   61      44   62    2.0                                 Trichosporon beigelii                                                                        C.I.    57      1.3  2.1   1.5                                 Saccharomyces cerevisiae                                                                     C.I.    41      67   53    1.9                                 ______________________________________                                         C.I. = Clinical isolate                                                  

                  TABLE 70                                                        ______________________________________                                        HYBRIDIZATION OF FUNGAL PROBES 1-4 TO RNA                                     OF ORGANISMS FOUND IN URINE                                                                      % Probe Bound                                              Organism      ATCC#      #1    #2    #3  #4                                   ______________________________________                                        Citrobacter freundii                                                                         8090      1.5   1.7   1.5 2.1                                  Enterobacter aerogenes                                                                      13048      2.5   1.9   2.0 2.0                                  Enterobacter cloacae                                                                        13047      2.5   1.6   2.6 2.0                                  Escherichia coli                                                                            11775      3.0   2.0   1.6 1.5                                  Klebsiella oxytoca                                                                          13182      2.5   2.2   2.5 2.0                                  Klebsiella pneumoniae                                                                       13883      2.5   2.2   2.1 2.0                                  Morganella morganii                                                                         25830      2.0   2.8   1.7 1.9                                  Proteus mirabilis                                                                           29906      2.5   1.9   2.3 2.0                                  Proteus vulgaris                                                                            13315      2.0   2.2   2.0 1.5                                  Providencia stuartii                                                                        29914      3.0   1.7   2.8 2.0                                  Pseudomonas aeruginosa                                                                      10145      2.0   1.9   1.3 2.0                                  Pseudomonas fluorescens                                                                     13525      2.5   2.7   2.1 2.0                                  Serratia marcescens                                                                         13880      2.5   1.7   1.8 2.0                                  Staphylococcus aureus                                                                       12600      2.0   1.7   1.8 2.0                                  Staphylococcus epidermidis                                                                  14990      3.0   1.5   1.3 2.0                                  Streptococcus agalactiae                                                                    13813      2.5   1.9   1.3 2.5                                  Streptococcus faecalis                                                                      19433      1.7   3.3   3.5 1.9                                  Streptococcus faecium                                                                       19434      2.0   2.9   2.1 1.5                                  Ureaplasma urealyticum                                                                      27618      2.1   3.1   2.4 1.8                                  ______________________________________                                    

                  TABLE 71                                                        ______________________________________                                        HYBRIDIZATION OF FUNGAL PROBES 1-4 To RNAs OF A CROSS                         SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS                                                    % Probe Bound                                              Organism       ATCC#     #1    #2    #3  #4                                   ______________________________________                                        Acinetobacter calcoaceticus                                                                  23055     2.5   2.5   2.0 1.9                                  Bacillus subtilis                                                                             6051     2.0   2.8   2.4 2.4                                  Bacteroides fragilis                                                                         23745     2.0   2.2   2.5 2.3                                  Branhamella catarrhalis                                                                      25238     2.5   3.2   1.8 1.7                                  Campylobacter jejuni                                                                         33560     2.5   2.1   2.0 1.9                                  Chlamydia trachomatis                                                                        VR878     3.1   3.1   1.8 2.7                                  Chromobacterium violaceum                                                                    29094     2.5   1.7   2.0 2.2                                  Clostridium perfringens                                                                      13124     1.9   2.3   1.8 1.8                                  Corynebacterium xerosis                                                                       373      1.6   4.8   1.8 1.1                                  Deinococcus radiodurans                                                                      35073     2.0   1.6   2.1 0.8                                  Derxia gummosa 15994     3.0   1.5   1.7 1.8                                  Gardnerella vaginalis                                                                        14018     2.0   2.2   1.3 1.2                                  Hafnia alvei   13337     1.0   2.5   1.7 1.6                                  Lactobacillus acidophilus                                                                     4356     2.0   2.7   2.0 1.9                                  Moraxella osloensis                                                                          19976     2.0   2.1   1.9 1.8                                  Mycobacterium smegmatis                                                                      14468     1.6   1.8   1.8 1.7                                  Mycoplasma hominis                                                                           14027     1.5   1.8   1.6 1.5                                  Neisseria gonorrhoeae                                                                        19424     2.0   2.7   1.6 1.6                                  Rahnella aquatilis                                                                           33071     2.0   2.7   2.3 2.1                                  Rhodospirillum rubrum                                                                        11170     2.0   1.8   1.6 1.5                                  Vibrio parahaemolyticus                                                                      17802     2.5   3.1   1.7 1.6                                  Yersinia enterocolitica                                                                       9610     2.0   1.8   2.3 2.2                                  Human                    2.0   1.8   2.1 3.0                                  ______________________________________                                    

Two derivatives of probe 1 also were made:

    CCCGACCGTCCCTATTAATCATTACGATGGTCCTAGAAAC

    CCCGACCGTCCCTATTAATCATTACGATGG

The first derivative works well at 65° C., the second at 60° C.

EXAMPLE 21

Gonorrhea is one of the most commonly reported bacterial infections inthe United States, with over two million cases reported annually. Thissexually transmitted disease usually results in anterior urethritis inmales and involves the cervix in females. While severe complications andeven sterility can occur in untreated individuals, asymptomaticinfections are common, resulting in carriers who unknowingly spread thedisease.

The causative agent, Neisseria gonorrhoeae, is a gram negative, oxidasepositive diplococcus with stringent growth requirements. The method usedfor diagnosis depends on the site of infection and the patient symptoms.Gonococcal urethritis in males is diagnosed with good sensitivity andspecificity using gram stain. Culture, requiring 24-72 hours, usuallymust be performed to confirm diagnosis of gonorrhea from all females andasymptomatic males. Following the detection of the organism from growthin culture, Neisseria gonorrhoeae must be identified by further testssuch as carbohydrate degradation, coagglutination, fluorescent antibodyscreens or chromogenic enzyme substrate assays.

Neisseria gonorrhoeae particularly difficult to detect and distinguishusing a nucleic said probe because it is very closely related to N.meningtidis. Data published in Kingsbury, D. T., J. Bacteriol.94:870-874 (1967) shows a DNA:DNA homology for the two species ofapproximately 80-94%. Under guidelines established by the Ad HocCommittee on Reconciliation of Approaches to Bacterial Systematics,Int'l J. System. Bacteriol. 37:463-464 (1987), the phylogeneticdefinition of a species generally means 70% or greater DNA:DNA homology.Despite the fact that these organisms may be considered to be the samespecies under established principles, we were able to make probescapable of distinguising them.

As expected, the rRNA homology between N. gonrrhoeae and N. meningitidisis even greater because of known conserved regions. We noted a 1.0%difference between the 16S and a 1.1% difference between the 23S rRNAsequences of N. gonorrhoeae and N. meningitidis using our sequencingdata.

Making a probe for N. gonorrhoeae was complicated by the fact that insome sites where N. meningitidis and N. gonorrhoeae differed, otherNeisseria species were similar to N. gonorrhoeae. The few mismatcheswhich exist between these two species are in the most variable regions,i.e., regions which vary not only between species, but also from strainto strain. Despite the fact that some believed the species could not bedistinguished with nucleic acid probes at all, and others believed thatrRNA was too conserved to be useful in probe diagnostics, we were ableto make probes capable of differentiating N. gonorrhoeae and N.meningitidis.

The present invention has significant advantages over each of the priorart methods; the probes are more specific and much faster than culturemethods. It also is believed that the probes are more sensitive, (i.e.,able to detect a smaller number of organisms in a clinical sample) thanprior art methods.

The primers used to identify these probe sequences had the followingsequences:

1. GGCCGTTACCCCACCTACTAGCTAAT

2. GTATTACCGCGGCTGCTGGCAC

3. GCTCGTTGCGGGACTTAACCCACCAT

Each of the rRNA sites chosen to target had at least two mismatches toE. coli, N. meningitidis, N. cinerea, N. lactamica, N. mucosa, andKingella kingae.

Oligonucleotides complementary to sequences adjacent to the proberegions were synthesized and used in the hydridization mix according toHogan et al., U.S. patent application Ser. No. 124,975, issued as U.S.Pat. No. 5,030,557, on Jul. 9, 1991, entitled "Means and Method forEnhancing Nucleic Acid Hybridization (the "helper" patent application).

The following sequences were characterized and shown to be specific forNeisseria gonorrhoeae. The phylogenetically nearest neighbors Neisseriameningtidis, N. lactamica, N. cinerea, N. mucosa, and Kingella kingaewere used for comparison with the N. gonorrhoeae sequence.

1. CCG CCG CTA CCC GGT AC

2. TCA TCG GCC GCC GAT ATT GGC

3. GAG CAT TCC GCA CAT GTC AAA ACC AGG TA

Sequence 1, complementary to 16S rRNA in the region 125-150, is 17 basesin length and has a Tm of 56° C. Sequence 2, complementary to 16S rRNAin the region 455-485, is 21 bases in length and has a Tm of 63° C.Sequence 3, complementary to 16S rRNA in the region 980-1015, is 29bases in length and has a Tm of 57° C.

The reactivity and specificity of the probes for Neisseria gonorrhoeaewas demonstrated with a hybridization assay. The three oligonucleotideprobes were iodinated and mixed with unlabeled oligonucleotides ofsequence 5'-CCC CTG CTT TCC CTC TCT AGA CGT ATG CGG TAT TAG CTG ATC TTTCG-3', 5'-GCC TTT TCT TCC CTG ACA AAA GTC CTT TAC AAC CCG-3', 5'-GGC ACGTAG TTA GCC GGT GCT TAT TCT TCA GGT AC-3', and 5'-GGT TCT TCG CGT TGCATC GAA TTA ATC CAC ATC ATC CAC CGC-3', and with purified RNA in 0.48Msodium phosphate, ph 6.8, 0.5% sodium dodecyl sulfate (SDS) andincubated at 60° C. for one hour. Following incubation, 4 ml of 2%hydroxyapatite, 0.12M sodium phosphate pH 6.8, 0.02% SDS was added andthe mixture was incubated at 60° C. for 5 minutes. The samples werecentrifuged and the supernatants were removed. Five ml of wash solution(0.12M sodium phosphate pH 6.8S, 2% SDS) was added and the samples weremixed, centrifuged, and the supernatants removed. The amount ofradioactivity bound to the hydroxyapatite was determined in a gammacounter.

Table 72 shows that the probes hybridize well to N. gonorrhoeae RNA anddo not hybridize to the other species tested.

                  TABLE 72                                                        ______________________________________                                        HYBRIDIZATION OF NEISSERIA GONORRHOEAE                                        PROBES 1-3 TO NEISSERIA AND KINGELLA RNAS                                     Organisms        ATCC #   % Probe Bound                                       ______________________________________                                        Kingella kingae  23332    0.09                                                Neisseria cinerea                                                                              14685    0.04                                                N. gonorrhoeae   19424    48.4                                                N. lactamica     23970    0.07                                                N. meningitidis serogroup A                                                                    13077    0.04                                                N. meningitidis serogroup B                                                                    13090    0.04                                                N. meningitidis serogroup C                                                                    13102    0.04                                                N. mucosa        19696    0.07                                                N. subflava      14799    0.05                                                ______________________________________                                    

The following derivatives of Neisseria probes also have been made andused:

    ______________________________________                                        GAG GAT TCC GCA CAT GTC AAA ACC AGG                                           GAG GAT TCC GCA CAT GTC AAA ACC AGG TAA                                       CCC GCT ACC CGG TAC GTT C                                                     CCG CTA CCC GGT ACG TTC.                                                      ______________________________________                                    

Although the above examples of performance were determined using thestandard assay format previously described, the specific probes may beused under a wide variety of experimental conditions. For example,additives may be included to the reaction solutions to provide optimalreaction conditions for accelerated hybridization. Such additives mayinclude buffers, chelators, organic compounds and nucleic acidprecipitating agents such as detergents, dihydroxybenzene, sodiumdodecyl sulfate, sodium diisobutyl sulfosuccinate, sodium tetradecylsulfate, sarkosyl and the alkali metal salts and ammonium salts of SO₄²⁺, PO₄ ³⁻, Cl⁻ and HCOO⁻. Such additives can be utilized by one skilledin the art to provide optimal conditions for the hybridization reactionto take place. These conditions for accelerated hybridization of singlestranded nucleic acid molecules into double stranded molecules are thesubject of the above-noted U.S. Pat. No. 5,132,207.

The present invention can be carried out on nonvital organisms frompurified samples or unpurified clinical samples such as sputum, feces,tissue, blood, spinal or synovial fluids serum, urine or other bodilyfluids, or other samples such as environmental or food samples. Prior tocell breakage and hybridization, the cells can be suspended or placed insolution. In the case of the unpurified samples referred to above, thecells may remain intact and untreated in their own biologicalenvironment prior to the assay.

The probes of the present invention may be used in an assay either aloneor in combination with different probes. Several individual probes alsocan be linked together during nucleic acid synthesis. This results inone probe molecule which contains multiple probe sequences, andtherefore, multiple specificities. For example, a single nucleic acidmolecule can be synthesized which contains both the Mycobacterium aviumand the Mycobacterium intracellulare sequences described in Examples 1and 2. When hybridized with either M.avium or M. intracellulare rRNAthis probe will hybridize completely. If the two probe sequences werecombined separately in an assay only one half of the mixed individualprobes will hybridize with either M.avium or M. intracellulare rRNA.Other embodiments also may be practiced within the scope of the claims.For example, probes may be labelled using a variety of labels, asdescribed within, and may be incorporated into diagnostic kits.

We claim:
 1. A probe comprising an oligonucleotide 10 to 100 nucleotidesin length able to hybridize to a Legionella nucleic acid target regionpresent in one or more Legionella species to form a detectabletarget:probe duplex under high stringency hybridization conditions, saidtarget region corresponding to, or fully complementary to and of thesame length as a nucleic acid corresponding to, a Legionella nucleicacid region selected from the group consisting of:bases 630-675 of E.coli 16S rRNA, bases 975-1020 of E. coli 16S rRNA, bases 350-395 of E.coli 23S rRNA, bases 1585-1620 of E. coli 23S rRNA, and bases 2280-2330of E. coli 23S rRNA;wherein said oligonucleotide comprises a segment of10 contiguous bases which is at least 75% complementary to a nucleotidesequence of 10 contiguous nucleotides present in a sequence selectedfrom the group consisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 6

and the sequences fully complementary and of the same length thereto;wherein said oligonucleotide does not hybridize to nucleic acid fromEscherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus andAcinetobacter calcoaceticus to form a detectable non-target:probe duplexunder said hybridization conditions.
 2. The probe of claim 1, whereinsaid one or more Legionella species is selected from the groupconsisting of: Legionella anisa, Legionella bozemanii, Legionellacherrii, Legionella dumoffii, Legionella erythra, Legionella feeleii,Legionella hackeliae, Legionella jamestowniensis, Legionella jordanis,Legionella longbeachea, Legionella maceachernii, Legionella micdadei,Legionella oakridgensis, Legionella parisiensis, Legionella pneumophila1, Legionella pneumophila 2, Legionella pneumophila 3, Legionellapneumophila 4, Legionella pneumophila 5, Legionella pneumophila 6,Legionella pneumophila 7, Legionella pneumophila 8, Legionellapneumophila 11, Legionella rubrilucens, Legionella sainthelensi,Legionella sainticrucis, Legionella spiritensis, Legionellasteigerewaltii, and Legionella wadsworthii.
 3. The probe of claim 2,wherein said oligonucleotide comprises a sequence selected from thegroup consisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 6

and the sequences fully complementary and of the same length thereto. 4.The probe of claim 2, wherein said target region corresponds to, or isfully complementary to and of the same length as a nucleic acidcorresponding to, bases 630-675 of E. coli 16S rRNA, and said nucleotidesequence of 10 contiguous nucleotides is present in either

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

or the sequence fully complementary and of the same length thereto. 5.The probe of claim 4, wherein said target region corresponds to bases630-675 of E. coli 16S rRNA and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC                   SEQ ID NO:
 1.


6. The probe of claim 2, wherein said target region corresponds to, oris fully complementary to and of the same length as a nucleic acidcorresponding to, bases 975-1020 of E. coli 16S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

or the sequence fully complementary and of the same length thereto. 7.The probe of claim 6, wherein said target region corresponds to bases975-1020 of E. coli 16S rRNA and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG                   SEQ ID NO:
 3.


8. The probe of claim 2, wherein said target region corresponds to, oris fully complementary to and of the same length as a nucleic acidcorresponding to, bases 350-395 of E. coli 23S rRNA, and said nucleotidesequence of 10 contiguous nucleotides is present in either

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC                 SEQ ID NO: 2

or the sequence fully complementary and of the same length thereto. 9.The probe of claim 8, wherein said target region corresponds to bases350-395 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC                 SEQ ID NO:
 2.


10. The probe of claim 2, wherein said target region corresponds to, oris fully complementary to and of the same length as a nucleic acidcorresponding to, bases 1585-1620 of E. coli 23S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC                            SEQ ID NO: 4

or the sequence fully complementary and of the same length thereto. 11.The probe of claim 10, wherein said target region corresponds to bases1585-1620 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC                            SEQ ID NO:
 4.


12. The probe of claim 2, wherein said target region corresponds to, oris fully complementary to and of the same length as a nucleic acidcorresponding to, bases 2280-2330 of E. coli 23S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

or the sequence fully complementary and of the same length thereto. 13.The probe of claim 12, wherein said target region corresponds to bases2280-2330 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    GTACCGAGGGTACCTTTGTGCT                                     SEQ ID NO:
 5.


14. The probe of claim 2, wherein said target region corresponds to, oris fully complementary to and of the same length as a nucleic acidcorresponding to, bases 2280-2330 of E. coli 23S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 6

or the sequence fully complementary and of the same length thereto. 15.The probe of claim 14, wherein said target region corresponds to bases2280-2330 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    CACTCTTGGTACGATGTCCGAC                                     SEQ ID NO:
 6.


16. The probe of any of claims 4-15, wherein said oligonucleotidecomprises a segment of 10 contiguous bases which is at least 90%complementary to said nucleotide sequence of 10 contiguous nucleotides.17. The probe of claim 16, wherein said oligonucleotide comprises asegment of 10 contiguous bases which is 100% complementary to saidnucleotide sequence of 10 contiguous nucleotides.
 18. The probe of claim16, wherein said oligonucleotide is 15 to 50 bases in length.
 19. Theprobe of claim 17, wherein said high stringency hybridization conditionscomprise 0.12M phosphate buffer containing equimolar amounts of Na₂ HPO₄and NaH₂ PO₄, 1 mM EDTA and 0.02% sodium dodecyl sulfate at 65° C.
 20. Aprobe comprising an oligonucleotide 15 to 100 nucleotides in length ableto hybridize to a Legionella nucleic acid target region present in oneor more Legionella species to form a detectable target:probe duplexunder high stringency hybridization conditions, said target regioncorresponding to, or fully complementary to and of the same length as anucleic acid corresponding to, a Legionella nucleic acid region selectedfrom the group consisting of:bases 630-675 of E. coli 16S rRNA, bases975-1020 of E. coli 16S rRNA, bases 350-395 of E. coli 23S rRNA, bases1585-1620 of E. coli 23S rRNA, and bases 2280-2330 of E. coli 23SrRNA;wherein said oligonucleotide comprises a segment of 15 contiguousbases which is at least 75% complementary to a nucleotide sequence of 15contiguous nucleotides present in a sequence selected from the groupconsisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 6

and the sequences fully complementary and of the same length thereto;wherein said oligonucleotide does not hybridize to nucleic acid fromEscherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus andAcinetobacter calcoaceticus to form a detectable non-target:probe duplexunder said hybridization conditions.
 21. The probe of claim 20, whereinsaid target region corresponds to a region selected from the groupconsisting of:bases 630-675 of E. coli 16S rRNA, bases 975-1020 of E.coli 16S rRNA, bases 350-395 of E. coli 23S rRNA, bases 1585-1620 of E.coli 23S rRNA, and bases 2280-2330 of E. coli 23S rRNA, and saidnucleotide sequence of 15 contiguous nucleotides is present in anucleotide sequence fully complementary to and of the same length as asequence selected from the group consisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

    and

    CACTCTTGGTACGATGTCCGAC                                     SEQ ID NO:
 6.


22. The probe of claims 20 or 21, wherein said oligonucleotide comprisesa segment of 15 contiguous bases which is at least 90% complementary tosaid nucleotide sequence of 15 contiguous nucleotides.
 23. The probe ofclaim 22, wherein said oligonucleotide comprises a segment of 15contiguous bases which is 100% complementary to said nucleotide sequenceof 15 contiguous nucleotides.
 24. The probe of claim 22, wherein saidoligonucleotide is 15-50 bases in length.
 25. A method of detectingwhether one or more Legionella species may be present in a samplecomprising the steps of:(a) contacting said sample with anoligonucleotide probe able to hybridize to a Legionella nucleic acidtarget region present in said one or more Legionella species to form adetectable target:probe duplex under high stringency hybridizationconditions, said target region corresponding to, or fully complementaryto and of the same length as a nucleic acid corresponding to, aLegionella nucleic acid region selected from the group consisting of:bases 630-675 of E. coli 16S rRNA, bases 975-1020 of E. coli 16S rRNA,bases 350-395 of E. coli 23S rRNA, bases 1585-1620 of E. coli 23S rRNA,and bases 2280-2330 of E. coli 23S rRNA;wherein said oligonucleotidecomprises a segment of 10 contiguous bases which is at least 75%complementary to a nucleotide sequence of 10 contiguous nucleotidespresent in a sequence selected from the group consisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 1

and the sequences fully complementary and of the same length thereto;wherein said oligonucleotide does not hybridize to nucleic acid fromEscherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus andAcinetobacter calcoaceticus to form a detectable non-target:probe duplexunder said hybridization conditions; and b) detecting hybridization ofsaid oligonucleotide to nucleic acid present in said sample under saidhybridization conditions as an indication that one or more Legionellaspecies may be present.
 26. The method of claim 25, wherein said one ormore Legionella species is selected from the group consisting of:Legionella anisa, Legionella bozemanii, Legionella cherrii, Legionelladumoffii, Legionella erythra, Legionella feeleii, Legionella hackeliae,Legionella jamestowniensis, Legionella jordanis, Legionella longbeachea,Legionella maceachernii, Legionella micdadei, Legionella oakridgensis,Legionella parisiensis, Legionella pneumophila 1, Legionella pneumophila2, Legionella pneumophila 3, Legionella pneumophila 4, Legionellapneumophila 5, Legionella pneumophila 6, Legionella pneumophila 7,Legionella pneumophila 8, Legionella pneumophila 11, Legionellarubrilucens, Legionella sainthelensi, Legionella sainticrucis,Legionella spiritensis, Legionella steigerewaltii, and Legionellawadsworthii.
 27. The method of claim 26, wherein said oligonucleotidecomprises a sequence selected from the group consisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 6

and sequences fully complementary and of the same length thereto. 28.The method of claim 26, wherein said target region corresponds to, or isfully complementary to and of the same length as a nucleic acidcorresponding to, bases 630-675 of E. coli 16S rRNA, and said nucleotidesequence of 10 contiguous nucleotides is present in either

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

or the sequence fully complementary and of the same length thereto. 29.The method of claim 28, wherein said target region corresponds to bases630-675 of E. coli 16S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC                   SEQ ID NO:
 1.


30. The method of claim 26, wherein said target region corresponds to,or is fully complementary to and of the same length as a nucleic acidcorresponding to, bases 975-1020 of E. coli 16S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

or the sequence fully complementary and of the same length thereto. 31.The method of claim 30, wherein said target region corresponds to bases975-1020 of E. coli 16S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG                   SEQ ID NO:
 3.


32. The method of claim 26, wherein said target region corresponds to,or is fully complementary to and of the same length as a nucleic acidcorresponding to, bases 350-395 of E. coli 23S rRNA, and said nucleotidesequence of 10 contiguous nucleotides is present in either

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

or the sequence fully complementary and of the same length thereto. 33.The method of claim 32, wherein said target region corresponds to bases350-395 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO:
 2.


34. The method of claim 26, wherein said target region corresponds to,or is fully complementary to and of the same length as to a nucleic acidcorresponding to, bases 1585-1620 of E. coli 23S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC                            SEQ ID NO: 4

or the sequence fully complementary and of the same length thereto. 35.The method of claim 34, wherein said target region corresponds to bases1585-1620 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC                            SEQ ID NO:
 4.


36. The method of claim 26, wherein said target region corresponds to,or is fully complementary to and of the same length as a nucleic acidcorresponding to, bases 2280-2330 of E. coli 23S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

or the sequence fully complementary and of the same length thereto. 37.The method of claim 36, wherein said target region corresponds to bases2280-2330 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    GTACCGAGGGTACCTTTGTGCT                                     SEQ ID NO:
 5.


38. The method of claim 26, wherein said target region corresponds to,or is fully complementary to and of the same length as a nucleic acidcorresponding to, bases 2280-2330 of E. coli 23S rRNA, and saidnucleotide sequence of 10 contiguous nucleotides is present in either

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 6

or the sequence fully complementary and of the same length thereto. 39.The method of claim 38, wherein said target region corresponds to bases2280-2330 of E. coli 23S rRNA, and said nucleotide sequence of 10contiguous nucleotides is present in the sequence fully complementaryand of the same length to

    CACTCTTGGTACGATGTCCGAC                                     SEQ ID NO:
 6.


40. The method of any of claims 28-39, wherein said oligonucleotidecomprises a segment of 10 contiguous bases which is at least 90%complementary to said nucleotide sequence of 10 contiguous nucleotides.41. The method of claim 40, wherein said oligonucleotide comprises asegment of 10 contiguous bases which is 100% complementary to saidnucleotide sequence of 10 contiguous nucleotides.
 42. The method ofclaim 40, wherein said oligonucleotide is 15-50 bases in length.
 43. Amethod of detecting whether one or more Legionella species may bepresent in a sample comprising the steps of:(a) contacting said samplewith a probe mix comprising three different oligonucleotide probes eachof which hybridizes to a first, a second, or a third Legionella nucleicacid target region present in said one or more Legionella species toform a first, a second, or a third detectable target:probe duplex underhigh stringency hybridization conditions, said first, said second, andsaid third, target region independently corresponding to, or fullycomplementary to and of the same length as a nucleic acid correspondingto, a Legionella nucleic acid region selected from the group consistingof: bases 630-675 of E. coli 16S rRNA, bases 975-1020 of E. coli 16SrRNA, bases 350-395 of E. coli 23S rRNA, bases 1585-1620 of E. coli 23SrRNA, and bases 2280-2330 of E. coli 23S rRNA; wherein each of saidthree different oligonucleotide probes independently comprises a segmentof 10 contiguous bases which is at least 75% complementary to anucleotide sequence of 10 contiguous nucleotides present in a sequenceselected from the group consisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

    CACTCTTGGTACGATGTCCGAC,                                    SEQ ID NO: 6

and the sequences fully complementary and of the same length thereto;wherein each of said three different oligonucleotide probes do nothybridize to nucleic acid from Escherichia coli, Pseudomonas aeruginosa,Vibrio parahaemolyticus and Acinetobacter calcoaceticus to form adetectable non-target:probe duplex under said hybridization conditions;and b) detecting whether said first, second, or said third probe:targetduplex has formed as an indication that said one or more Legionellaspecies may be present in said sample, wherein said three differentoligonucleotide probes together result in the formation of at least onedetectable probe:target duplex with nucleic acid present in each ofLegionella anisa, Legionella bozemanii, Legionella cherrii, Legionelladumoffii, Legionella erythra, Legionella feeleli, Legionella hackeliae,Legionella jamestowniensis, Legionella jordanis, Legionella longbeachea,Legionella maceachernii, Legionella micdadei, Legionella oakridgensis,Legionella parisiensis, Legionella pneumophila 1, Legionella pneumophila2, Legionella pneumophila 3, Legionella pneumophila 4, Legionellapneumophila 5, Legionella pneumophila 6, Legionella pneumophila 7,Legionella pneumophila 8, Legionella pneumophila 11, Legionellarubrilucens, Legionella sainthelensi, Legionella sainticrucis,Legionella spiritensis, Legionella steigerewaltii, and Legionellawadsworthii.
 44. The probe of claim 43, wherein said first, said second,and said third target region independently corresponds to a regionselected from the group consisting of:bases 630-675 of E. coli 16S rRNA,bases 975-1020 of E. coli 16S rRNA, bases 350-395 of E. coli 23S rRNA,bases 1585-1620 of E. coli 23S rRNA, and bases 2280-2330 of E. coli 23SrRNA;and said nucleotide sequence of 10 contiguous nucleotides ispresent in a nucleotide sequence fully complementary to and of the samelength as a sequence selected from the group consisting of:

    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC,                  SEQ ID NO: 1

    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC,                SEQ ID NO: 2

    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG,                  SEQ ID NO: 3

    GCGGTACGGTTCTCTATAAGTTATGGCTAGC,                           SEQ ID NO: 4

    GTACCGAGGGTACCTTTGTGCT,                                    SEQ ID NO: 5

and

    CACTCTTGGTACGATGTCCGAC                                     SEQ ID NO:
 6.


45. The method of claim 43, wherein each of said three differentoligonucleotide probes independently comprises a segment of 10contiguous bases which is at least 90% complementary to said nucleotidesequence of 10 contiguous nucleotides.
 46. The method of claim 45,wherein each of said three different oligonucleotide probesindependently comprises a segment of 10 contiguous bases which 100%complementary to said nucleotide sequence of 10 contiguous nucleotides.47. The method of any of claims 43-46 wherein said first and said seconddetectable probe:target duplex is formed with a different target region.48. The method of any of claims 43-46 wherein said first, said second,and said third detectable probe:target duplex is formed with a differenttarget region.